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Quantitative analysis of LP-BM5 murine leukemia retrovirus RNA using real-time RT-PCR
W James Cook1, Kathy A Green, Joshua J Obar
1Department of Microbiology and Immunology, Dartmouth Medical School and the Norris Cotton Cancer Center, Borwell Building, One Medical Center Drive, Lebanon, NH 03756, USA.
Abstract:
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.
Insights
New quantitative assays help researchers understand Murine AIDS (MAIDS) development. These tools track defective (BM5def) and helper (BM5eco) viruses, revealing CD40
Area of Science:
- Virology
- Immunology
- Molecular Biology
Background:
- Murine AIDS (MAIDS) is induced by the LP-BM5 murine leukemia virus (MuLV) complex, comprising defective (BM5def) and replication-competent (BM5eco) viruses.
- BM5def is the primary cause of MAIDS, while BM5eco acts as a helper virus essential for BM5def propagation.
- Understanding the distinct roles and quantities of these viral components is crucial for MAIDS pathogenesis research.
Purpose of the Study:
- To develop and validate quantitative real-time RT-PCR assays for quantifying BM5def and BM5eco RNA.
- To standardize LP-BM5 virus preparations for consistent experimental dosing.
- To investigate the viral RNA levels in MAIDS-susceptible versus CD40-deficient MAIDS-insusceptible mice.
Main Methods:
- Development of quantitative real-time reverse transcription PCR (RT-PCR) assays.
- Quantification of BM5def and BM5eco gag RNA in spleen and blood cells.
- Comparison of viral RNA levels between MAIDS-susceptible and CD40-deficient mice.
Main Results:
- BM5def gag RNA levels were similar in spleens of both MAIDS-susceptible and CD40-deficient mice.
- BM5eco gag RNA levels were significantly higher (up to 10-fold) in spleens of CD40-deficient mice compared to controls.
- Similar trends were observed in blood, with similar BM5def RNA but increased BM5eco RNA in CD40-deficient mice.
Conclusions:
- The developed RT-PCR assays enable precise quantification of LP-BM5 viral components.
- CD40 deficiency influences the propagation of the helper virus (BM5eco) but not the causative agent (BM5def).
- These findings facilitate a better understanding of the differential contributions of viral components to MAIDS pathogenesis.