Gene expression profiling of microglia infected by a highly neurovirulent murine leukemia virus: implications for neuropathogenesis.
BACKGROUND
Certain murine leukemia viruses (MLVs) are capable of inducing progressive spongiform motor neuron disease in susceptible mice upon infection of the central nervous system (CNS). The major CNS parenchymal target of these neurovirulent retroviruses (NVs) are the microglia, whose infection is largely coincident with neuropathological changes. Despite this close association, the role of microglial infection in disease induction is still unknown. In this paper, we investigate the interaction of the highly virulent MLV, FrCasE, with microglia ex vivo to evaluate whether infection induces specific changes that could account for neurodegeneration. Specifically, we compared microglia infected with FrCasE, a related non-neurovirulent virus (NN) F43/Fr57E, or mock-infected, both at a basic virological level, and at the level of cellular gene expression using quantitative real time RT-PCR (qRT-PCR) and Afffymetrix 430A mouse gene chips.


RESULTS
Basic virological comparison of NN, NV, and mock-infected microglia in culture did not reveal differences in virus expression that provided insight into neuropathogenesis. Therefore, microglial analysis was extended to ER stress gene induction based on previous experiments demonstrating ER stress induction in NV-infected mouse brains and cultured fibroblasts. Analysis of message levels for the ER stress genes BiP (grp78), CHOP (Gadd153), calreticulin, and grp58 in cultured microglia, and BiP and CHOP in microglia enriched fractions from infected mouse brains, indicated that FrCasE infection did not induce these ER stress genes either in vitro or in vivo. To broadly identify physiological changes resulting from NV infection of microglia in vitro, we undertook a gene array screen of more than 14,000 well-characterized murine genes and expressed sequence tags (ESTs). This analysis revealed only a small set of gene expression changes between infected and uninfected cells (<18). Remarkably, gene array comparison of NN- and NV-infected microglia revealed only 3 apparent gene expression differences. Validation experiments for these genes by Taqman real-time RT-PCR indicated that only single Ig IL-1 receptor related protein (SIGIRR) transcript was consistently altered in culture; however, SIGIRR changes were not observed in enriched microglial fractions from infected brains.


CONCLUSIONS
The results from this study indicate that infection of microglia by the highly neurovirulent virus, FrCasE, does not induce overt physiological changes in this cell type when assessed ex vivo. In particular, NV does not induce microglial ER stress and thus, FrCasE-associated CNS ER stress likely results from NV interactions with another cell type or from neurodegeneration directly. The lack of NV-induced microglial gene expression changes suggests that FrCasE either affects properties unique to microglia in situ, alters the expression of microglial genes not represented in this survey, or affects microglial cellular processes at a post-transcriptional level. Alternatively, NV-infected microglia may simply serve as an unaffected conduit for persistent dissemination of virus to other neural cells where they produce acute neuropathogenic effects.
Dimcheff
Derek E
DE
Laboratory of Persistent Viral Diseases, Rocky Mountain Laboratories, NIAID, NIH, Hamilton, MT, USA. derekdim@umich.edu
Volkert
L Gwenn
LG
Li
Ying
Y
DeLucia
Angelo L
AL
Lynch
William P
WP
D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D001921:Brain / Q000166:cytology; D018929:Cell Culture Techniques / Q000379:methods; D017931:DNA Primers; D000091342:Endoplasmic Reticulum Chaperone BiP; D020869:Gene Expression Profiling; D015683:Gene Products, gag / Q000235:genetics; D009052:Leukemia Virus, Murine / Q000235:genetics* / Q000472:pathogenicity*; D051379:Mice / Q000821:virology; D008815:Mice, Inbred Strains; D017628:Microglia / Q000502:physiology / Q000821:virology*; D009693:Nucleic Acid Hybridization; D020411:Oligonucleotide Array Sequence Analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014774:Virulence
D017931:DNA Primers; D000091342:Endoplasmic Reticulum Chaperone BiP; D015683:Gene Products, gag; C000717248:Hspa5 protein, mouse