Studies of the genomic RNA of leukosis viruses: implications for RNA dimerization

Abstract

Retroviral particles contain two positive-strand genomic RNAs linked together by noncovalent bonds that can be dissociated under mild conditions. We studied genomic RNAs of wild-type and mutant avian leukosis viruses (ALVs) in an attempt to (i) better understand the site(s) of RNA dimerization, (ii) examine whether the primer binding site (PBS) and tRNA primer are involved in dimerization, and (iii) determine the structure of genomic RNA in protease-deficient (PR(-)) mutants. We showed that extensively nicked wild-type ALV genomic RNAs melt cooperatively. This implies a complex secondary and/or tertiary structure for these RNAs that extends well beyond the 5' dimerization site. To investigate the role of the PBS-tRNA complex in dimerization, we analyzed genomic RNAs from mutant viruses in which the tRNA(Trp) PBS had been replaced with sequences homologous to the 3' end of six other chicken tRNAs. We found the genomic RNAs of these viruses are dimers that dissociate at the same temperature as wild-type viral RNA, which suggests that the identity of the PBS and the tRNA primer do not affect dimer stability. We studied two ALV PR(-) mutants: one containing a large (>1.9-kb) inversion spanning the 3' end of gag and much of pol, rendering it deficient in PR, reverse transcriptase, and integrase, and another with a point mutation in PR. In both of these mutant viruses, the genomic RNA appears to be either primarily or exclusively monomeric. These data suggest that ALV can package its RNA as monomers that subsequently dimerize.

FIG. 1

Northern blot showing thermal stability of genomic RNA from wild-type ALV/RCASBP(A) virus. Purified viral RNA was aliquoted and incubated at the indicated temperature for 10 min in standard R buffer. After fractionation on a 1% nondenaturing agarose gel, the RNA was transferred to a positively charged nylon membrane and hybridized with a ³²P-labeled riboprobe homologous to nucleotides 4993 to 5256 (taking the 5′ end of the genomic RNA as position 1). Arrows indicate the positions of migration of the 70S RNA dimer and 35S RNA monomer.

FIG. 2

Northern blot showing thermal stability of nicked genomic RNA from wild-type ALV/RCASBP(A) virus. Purified viral RNAs were resuspended in either standard R buffer or H buffer. Electrophoresis and Northern blot analysis were performed as described in the legend to Fig. 1.

FIG. 3

(A) Hypothetical model of RSV dimer linkage structure originally proposed by Haseltine et al. (23), based on the nucleotide sequence of RSV Pr-C strong-stop DNA. The diagram at the top shows potential base pairing between two antiparallel genomic RNA monomers and includes two tRNA^Trp primer molecules as part of the linkage. The tRNA primers are shaded in gray, PBSs are shown as white letters in black boxes, and the second-strand binding sites are enclosed by boxes. The scheme below shows base pairing between the U5 and PBS regions of the monomers in the absence of tRNA primers (redrawn, with permission, from reference 10). (B and C) Schematics of RSV SR-A derived wild-type RCASBP(A) and PBS mutants showing base pairing between a tRNA primer and each strand of genomic RNA. (B) Sequence of RSV SR-A derived RCASBP(A) wild-type PBS with the 3′ end of the tRNA^Trp annealed to it and also the altered PBS sequences in the mutant viruses. The white letters in black boxes signify the differences between the wild-type and mutant PBS sequences. The tRNA anticodon specificities are tRNA^Met(CAU), tRNA^Pro(AGG), tRNA^Lys(CUU), tRNA^Phe(GAA), tRNA^Ile(AAU), and tRNA^Ser(UCA). (These mutants were previously described in and the figure is redrawn from reference .) (C) Proposed base pairing between RSV SR-A-derived wild-type RCASBP(A) genomic RNA and the tRNA^Trp primer molecule of the second genomic RNA strand showing mispairing between alternatively specified tRNAs of the mutant viruses and the putative secondary binding site in genomic RNA. The white letters in black boxes signify the differences between the wild-type and mutant tRNA sequences.

FIG. 4

Northern blot showing thermal stability of genomic RNAs from ALV/RCASBP(A) PBS mutants RCASBP(A)(MetPBS), RCASBP(A)(PhePBS), and RCASBP(A)(IlePBS). Arrows indicate the positions of migration of the dimeric and monomeric RNAs. Melting was done in standard R buffer at the indicated temperatures as described in the legend to Fig. 1. PBS mutant constructs are described in Fig. 3.

FIG. 5

Diagram of genomic structures of wild-type ALV/RCAS(A) (RCAS-wt) and mutant proviruses. Mutant RCAS-AvrII has an inversion of the sequence between nucleotides 2435 and 4372, disrupting PR, RT, and IN. RCAS-ΔHpa/Kpn has a deletion of nucleotides 2734 to 4999 disrupting RT and IN. RCAS-ΔSal/Cla has a deletion of nucleotides 6057 to 7030 disrupting SU (surface) and TM (transmembrane) proteins. Nucleotide positions are given with the 5′ end of the genomic RNA taken as position 1 (these mutants were described in and the figure is redrawn from reference 19). Mutant RCASneoD37I is a point mutant in which the PR active-site aspartic acid at position 37 is changed to an isoleucine, rendering it PR⁻. LTR, long terminal repeat; MA, CA, and NC, matrix, capsid, and nucleocapsid, respectively.

FIG. 6

Northern blot showing thermal stability of genomic RNAs from wild-type ALV/RCAS(A) and two deletion mutants. Virions were isolated from transiently transfected CEFs, and viral RNAs were purified. Melting was done in standard R buffer at the indicated temperatures as described in the legend to Fig. 1. RCAS-ΔHpa/Kpn is RT⁻ and IN⁻, while RCAS-ΔSal/Cla is SU⁻ and TM⁻. Mutants are further described in Fig. 5.

FIG. 7

Northern blot showing thermal stability of genomic RNAs from wild-type ALV/RCAS(A) and two PR⁻ mutants. Virions were isolated from transiently transfected DF-1 cells, and viral RNAs were purified. Melting was done in standard R buffer at the indicated temperatures as described in the legend to Fig. 1. (A) Northern blot melting profiles of genomic RNAs from wild-type ALV/RCAS(A) (RCAS-wt) and mutant RCAS-AvrII. Mutant RCAS-AvrII is PR⁻, RT⁻, and IN⁻ and is described further in Fig. 5. (B) Northern blot melting profiles of genomic RNAs from wild-type RSV and mutant RCASneoD37I. Mutant RCASneoD37I has a point mutation at the active site rendering it PR⁻.

FIG. 8

Northern blot showing thermal stability of genomic RNAs from wild-type ALV/RCAS(A) (RCAS-wt) and mutants RCAS-AvrII and RCASneoD37I. Virions were isolated from transiently transfected DF-1 cells and lysed in high-salt (500 mM NaCl) buffer, and viral RNAs were purified. Melting was done at the indicated temperatures as described in the legend to Fig. 1 except that RNA was suspended in high-salt buffer. Arrows indicate positions of migration of the dimeric and monomeric RNAs. Mutants are further described in Fig. 5.