Replication and pathogenicity of primer binding site mutants of SL3-3 murine leukemia viruses

Abstract

Retroviral reverse transcription is primed by a cellular tRNA molecule annealed to an 18-bp primer binding site sequence. The sequence of the primer binding site coincides with that of a negatively acting cis element that mediates transcriptional silencing of murine leukemia virus (MLV) in undifferentiated embryonic cells. In this study we test whether SL3-3 MLV can replicate stably using tRNA primers other than the cognate tRNAPro and analyze the effect of altering the primer binding site sequence to match the 3' end of tRNA1Gln, tRNA3Lys, or tRNA1,2Arg in a mouse pathogenicity model. Contrary to findings from cell culture studies of primer binding site-modified human immunodeficiency virus type 1 and avian retroviruses, our findings were that SL3-3 MLV may stably and efficiently replicate with tRNA primers other than tRNAPro. Although lymphoma induction of the SL3-3 Lys3 mutant was significantly delayed relative to that of the wild-type virus, molecular tumor analysis indicated that all the primer binding site-modified viruses induce T-cell lymphomas similar to those induced by the wild-type virus in terms of frequencies of genomic rearrangements within the T-cell receptor beta-chain, the immunoglobulin kappa light chain, and the c-myc locus. Whereas none of the mutants were found to revert to tRNAPro primer utilization, in two tumors resulting from the injection of the SL3-3 Lys3 mutant the primer binding site was altered to match that of a new primer species, tRNA1,2Lys. In addition, recombination with endogenous viruses resulting in the generation of recombinant viruses carrying a glutamine primer binding site was detected in the majority of the tumors induced by the SL3-3 Lys3 mutant as well as in two tumors induced by wild-type SL3-3 and the SL3-3 Arg1,2 mutant.

FIG. 1

Lymphoma development in inbred NMRI mice injected with wt and mutated SL3-3 MLVs. The cumulative mortality is plotted as a function of time after virus injection. (A) PBS-modified SL3-3 viruses; (B) 5′ UTR-modified virus.

FIG. 2

Southern blot of tumor DNAs digested with HindIII and probed for rearrangements in their TCR β loci (J2 probe). The arrow indicates the position of the nonrearranged germ line band.

FIG. 3

(A) wt SL3-3 and mutant PBS-tRNA base pairings. Note that tRNA₂^Gln binding to the Gln1 PBS does not disrupt base pairing. (B) Potential base pairings between the Lys3 PBS and various tRNA species. Note that 14 base pairings may be formed between the Lys3 PBS and tRNA_1,2^Lys. Numbers to the right of the panels indicate numbers of bases paired.