Quantitative analysis of LP-BM5 murine leukemia retrovirus RNA using real-time RT-PCR
Introduction
LP-BM5 is a murine retrovirus isolate that causes a murine immunodeficiency syndrome in genetically susceptible mouse strains characterized by splenomegaly, lymphadenopathy, hypergammaglobulinemia, T- and B-cell immune dysfunctions, increased susceptibility to opportunistic infections, and late stage lymphomas (Buller et al., 1987, Cerny et al., 1990, Huang et al., 1995, Klinken et al., 1988, Klinman and Morse, 1989, Legrand et al., 1981, Morse et al., 1989, Morse et al., 1992, Mosier et al., 1987, Pattengale et al., 1982, Yetter et al., 1988). This murine leukemia virus (MuLV) mixture includes ecotropic (BM5eco), recombinant mink cell focus-inducing (BM5mcf), and replication-defective (BM5def) viruses, with the BM5def genome serving as the proximal agent causing the immunodeficiency (Aziz et al., 1989, Chattopadhyay et al., 1989, Hartley et al., 1989, Huang et al., 1989, Jolicoeur, 1991, Kubo et al., 1996, Mosier et al., 1985). Neither the replication-competent BM5eco nor BM5mcf viruses, alone or in combination, induce immunodeficiency but instead serve as helper viruses for replication of the defective BM5def genome (Chattopadhyay et al., 1989, Chattopadhyay et al., 1991).
Development of murine AIDS (MAIDS) symptoms following LP-BM5 infection is dependent on the mouse strain and integrity of the host immune system. Genetically susceptible C57BL/6 (B6) and B10 strains containing intact immune systems are highly susceptible, while other fully immunocompetent strains including A/J, BALB/c, and 129 are resistant (Hamelin-Bourassa et al., 1989, Hartley et al., 1989, Huang et al., 1992, Makino et al., 1990, Makino et al., 1992). Nude and SCID mutant mice on the B6 background are insusceptible to MAIDS symptoms (Green et al., 2001, Mosier et al., 1987, Simard et al., 1997), as are B6 mice lacking, among others, the CD40 or CD40L (CD154) genes (Green et al., 1996, Green et al., 1998, Green et al., 2001, Yu et al., 1999). Previous studies utilizing semi-quantitative RT-PCR assays showed that CD40-deficient (Yu et al., 1999) and CD154 antibody-neutralized (Green et al., 1996) mice harbor BM5def viral loads roughly comparable to susceptible B6 mice indicating that in these cases MAIDS resistance is not simply due to lack of early BM5def propagation.
Previous RT-PCR assays used to quantify BM5def (Giese et al., 1994, Green et al., 1996, Yu et al., 1999) and BM5eco (Gazzinelli et al., 1994, Lee et al., 1988) RNA were semi-quantitative. Functional measurement of helper viral load was restricted to XC plaque assays for BM5eco (Rowe et al., 1970) and foci-forming assays for BM5mcf (Hartley et al., 1977). To more quantitatively monitor RNA levels of the LP-BM5 components in viral inocula and infected mouse tissues, we developed real-time RT-PCR assays for replication-defective (BM5def) and -competent (BM5eco) gag RNAs. Our results show that in viral inocula and in spleen and blood of susceptible strains, BM5def gag RNA is present at higher levels than that of BM5eco. Our results also show that while BM5def gag RNA is present at roughly similar levels in spleen and blood of MAIDS-susceptible and -insusceptible CD40-deficient mice, BM5eco gag RNA is up to 10-fold more abundant in spleen and blood of CD40-deficient mice.
Section snippets
LP-BM5 RNA preparations and provirus clones
LP-BM5 was prepared as described previously (Klinken et al., 1988). To isolate viral RNA, LP-BM5 virions were purified by ultracentrifugation at 40,000 rpm for 30 min, followed by cell lysis and RNA purification using RNeasy columns with DNAse-I treatment (Qiagen, Valencia, CA) following the manufacturer's protocols. BM5 MCF#7 RNA was isolated from mink lung cells infected with G6 BM5 MCF#7 (Chattopadhyay et al., 1991; kind gift from Dr. Janet Hartley) using the RNeasy protocol. Provirus
Development of quantitative real-time RT-PCR assays for BM5def and BM5eco gag RNA
To compare quantitatively BM5def and BM5eco RNA levels in viral stocks and infected mouse tissues, we developed real-time RT-PCR assays to measure directly RNA levels of each viral component. Because expression of LP-BM5 gag RNA is likely to be regulated differently than that of pol or env due to post-transcriptional mechanisms including alternative RNA splicing (Coffin, 1996), we restricted the analysis to gag RNA. Primers for BM5def gag were described previously (Giese et al., 1994). Primers
Discussion
We describe the development of real-time RT-PCR assays to quantify RNA of the functional components of the LP-BM5 MuLV mixture. Real-time RT-PCR has been used to quantify viral load and viral gene expression in several viral systems (Brudevi and Weinstock, 2001, Lewin et al., 1999, Martell et al., 1999, Trottier et al., 2002). While the majority of reported real-time viral load assays use the more sensitive TaqMan or molecular beacon methods, these methods are often not feasible when DNA
Acknowledgements
We thank Herbert C. Morse and Janet Hartley for kindly providing the original stock of LP-BM5-infected G6 cells, Janet Hartley for kindly providing mink lung cells infected with BM5 MCF#7, Sisir Chattopadhyay for provirus clones, Cory Ahonen for providing the CD40 k.o. and EkoK2 transgenic mice, and Burkhard Becher and Sergio Quezada for assistance and advice on use of the iCycler iQ instrument. We also are appreciative of Randy Noelle, On Ho, Arti Gaur, Meghan Brennan, and Robert Rich for
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