Quantitative analysis of LP-BM5 murine leukemia retrovirus RNA using real-time RT-PCR.
Murine AIDS (MAIDS) develops in susceptible mouse strains after infection with the LP-BM5 murine leukemia virus (MuLV) complex that contains a mixture of defective (BM5def) and replication-competent viruses. While the BM5def virus is the causative agent in MAIDS, the replication-competent viruses in LP-BM5, including ecotropic MuLV (BM5eco), are required for BM5def propagation and thus function as helper viruses. We describe quantitative real-time RT-PCR assays for RNA encoded by the BM5def and BM5eco components of LP-BM5. The assays were used to standardize better the input doses of LP-BM5 viruses across viral preparations and to quantify BM5def and BM5eco gag RNA levels in spleen and blood cells from MAIDS-susceptible and -insusceptible infected mice. Spleens of MAIDS-susceptible infected mice harbored approximately similar levels of BM5def gag RNA as infected spleens of mice that are insusceptible to MAIDS due to lack of CD40. In contrast, the same infected spleens of CD40-deficient mice contained substantially higher (up to 10-fold) levels of BM5eco gag RNA compared with susceptible controls. Similar to that seen in spleen, infected blood of CD40-deficient mice contained similar levels of BM5def gag as susceptible strains, but increased levels (up to threefold) of BM5eco gag RNA. The assays described below can be used to characterize better the contributions of different functional viral components of the LP-BM5 mixture to the development of MAIDS.
Cook
W James
WJ
Department of Microbiology and Immunology, Dartmouth Medical School and the Norris Cotton Cancer Center, Borwell Building, One Medical Center Drive, Lebanon, NH 03756, USA.
Green
Kathy A
KA
Obar
Joshua J
JJ
Green
William R
WR
D000818:Animals; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D015870:Gene Expression; D015750:Genes, gag; D006378:Helper Viruses / Q000235:genetics; D009052:Leukemia Virus, Murine / Q000235:genetics* / Q000472:pathogenicity; D051379:Mice; D018345:Mice, Knockout; D008822:Mice, Transgenic; D016183:Murine Acquired Immunodeficiency Syndrome / Q000821:virology; D012367:RNA, Viral / Q000032:analysis* / Q000097:blood / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013154:Spleen / Q000821:virology; D014773:Virology / Q000379:methods*
D017931:DNA Primers; D012367:RNA, Viral