Recombination in the 5' leader of murine leukemia virus is accurate and influenced by sequence identity with a strong bias toward the kissing-loop dimerization region

Abstract

Retroviral recombination occurs frequently during reverse transcription of the dimeric RNA genome. By a forced recombination approach based on the transduction of Akv murine leukemia virus vectors harboring a primer binding site knockout mutation and the entire 5' untranslated region, we studied recombination between two closely related naturally occurring retroviral sequences. On the basis of 24 independent template switching events within a 481-nucleotide target sequence containing multiple sequence identity windows, we found that shifting from vector RNA to an endogenous retroviral RNA template during minus-strand DNA synthesis occurred within defined areas of the genome and did not lead to misincorporations at the crossover site. The nonrandom distribution of recombination sites did not reflect a bias for specific sites due to selection at the level of marker gene expression. We address whether template switching is affected by the length of sequence identity, by palindromic sequences, and/or by putative stem-loop structures. Sixteen of 24 sites of recombination colocalized with the kissing-loop dimerization region, and we propose that RNA-RNA interactions between palindromic sequences facilitate template switching. We discuss the putative role of the dimerization domain in the overall structure of the reverse-transcribed RNA dimer and note that related mechanisms of template switching may be found in remote RNA viruses.

FIG. 1

Alignment of Akv and MLEV 5′ UTRs. The sequence of MLEV was determined by various PCR-based methods as described previously (38) and in the text. PBS sequences are indicated in bold letters. Identical nucleotide positions are indicated by asterisks; nucleotide insertions in Akv and MLEV are indicated by introduction of colons (:) in MLEV and Akv sequences, respectively. Single-nucleotide differences and clusters of differences are underlined in the MLEV sequence, and the marker number (LM1 to LM34) is given below each genetic marker within the packaging region ranging from the 3′ PBS (position 163) to the gag start codon (position 638); LM1 to LM16 correspond to markers IV to XVIII in reference . SIWs between markers are indicated by brackets and the designations SIWI to SIWXX. Glyco-gag (positions 375 to 377) and gag (positions 639 to 641) start codons are given in italics. Palindromes (eight nucleotides or longer) in Akv are underlined. The lengths of Akv and MLEV 5′ UTR sequences are 476 and 465 bp, respectively; due to a total of five nucleotide insertions in MLEV (indicated by colons in Akv sequence), the length of the entire recombination window is 481 bp.

FIG. 2

Vector structure and function. (A) Vectors, expression in packaging cells, and transductional titer. A modified PBS sequence, designated PBS-Umu, was introduced in Akv MLV-derived vectors harboring the neo gene embedded in the bacterial Tn5 transposon. pPBSPro244ΨAkv-neo and pPBSUmu244ΨAkv-neo were previously used in studies of retroviral recombination (38). The entire Akv 5′ UTR was inserted in vectors pPBSPro476ΨAkv-neo and pPBSUmu476ΨAkv-neo. Vectors studied in this work and previously differ in the length of the 5′ UTR sequence, being 244 and 476 bp, respectively. neo expression was estimated by transfection of Ψ-2 packaging cells followed by counting of G418-resistant colonies; the resulting estimate of vector expression is given as 10² G418-resistant colonies per transfection of 10 μg of vector DNA. Transductional titers were measured by counting the G418-resistant CFU per milliliter of medium transferred from stably transfected packaging cells; titers have been normalized to 10⁷ producer cells and represent average values for three independent experiments. ND, not determined. The schematic representation of the secondary structure of the MLV 5′ UTR is based on studies by Tounekti et al. (62) and Mougel et al. (42). Ten putative stem-loops (SLs 1 to 10) are found within the region. As indicated by dotted lines, SLs 1 to 9 and a putative shortened SL 10 were included in the longer vectors harboring the complete 5′ UTR, whereas only SLs 1 to 4 were included in the shorter vectors (38). By analogy with the previously presented model, stem-loops are located as follows (position numbers as given in Fig. 1): SL 1, 236 to 265; SL 2, 291 to 322; SL 3, 329 to 370; SL 4, 372 to 394; SL 5, 400 to 417; SL 6, 423 to 453; SL 7, 459 to 480; SL 8, 486 to 534; SL 9, 539 to 561; and SL 10, 605 to 647. (B) Experimental approach for studies of forced recombination. PBS-mutated vectors were tested in single-cycle transfer protocol by transfection into Ψ-2 packaging cells and subsequent virus transfer to NIH 3T3 target cells. Recombination-based vector transduction, or forced recombination, is the result of (i) heterodimeric RNA encapsidation, (ii) initiation of minus-strand synthesis, (iii) successful plus-strand transfer, and (iv) expression of the neo gene. G418-resistant colonies were individually cloned or pooled (see Materials and Methods) in order to sequence individual transduced proviruses or to allow PCR screening for recombinants, respectively.

FIG. 3

Nucleotide sequences of PBS-Gln-harboring transduced proviruses and recombination partners Akv and MLEV. The sequences of individual transduced proviruses were determined by sequence analysis of PCR fragments encompassing the entire 5′ UTR (here defined as the region from PBS to gag start codon). Transduced viral sequences are compared with homologous regions of Akv (top) and MLEV (bottom). Two sequences, 28 and 42, originate from analysis of individual colony clones (Table 1); the remaining sequences originate from PCR screening of colony pools obtained from separate plates and subsequent sequence analysis. Nucleotides homologous to positions in Akv are indicated by hyphens; deleted nucleotides compared to Akv are indicated by colons, whereas insertions are indicated by introduction of a colon in the Akv sequence. Genetic markers consisting of more than one-nucleotide differences are underlined. Molecular differences between Akv-neo and MLEV within the 5′ UTR are designated LM1 through LM34; marker numbering is indicated below the Akv sequence. LM1 to LM16 correspond to markers IV to XVIII in reference . The gag start codon is indicated for convenience (position 639); however, the ATG sequence was not included in the vectors utilized. R, A/G mixed nucleotide position.

FIG. 4

Mapping of sites for recombinational repair of PBS-modified vectors. Template switching during minus-strand DNA synthesis within the 5′ UTR between the PBS and neo gene is established to obtain perfect PBS complementarity in second-strand transfer. Thin lines indicate RNA; thick lines represent DNA. The sloping line represents the 5′ UTR with genetic markers LM1 to LM34. Stippled dots indicate single-nucleotide differences, whereas clusters of differences are indicated by black dots. Lengths of SIWs are given between genetic markers. Recombination sites are dispersed throughout the region as delineated by arrow width and ratios (number of proviruses with specific recombination site/total number of proviruses analyzed). In 16 of 24 analyzed proviral sequences harboring PBS-Gln, the recombination site was mapped within a region coinciding with the kissing stem-loop dimerization domain. The kissing stem-loop (SL 2 [Fig. 2A]) harbors LM8 and LM9, whereas LM10 is located within SL 3 (Fig. 2A).

FIG. 5

Model for kissing-loop-mediated recombination (not drawn to scale). The interaction of Akv and MLEV kissing loops and putative subsequent RNA duplex formation promote template switching during viral DNA synthesis, most likely due to close RNA-RNA interactions in the region. Enlargements of the interacting Akv and MLEV stem-loops and the RNA duplex supposedly generated subsequent to loop kissing are shown. It should be noted that the interaction of kissing loops represents a local antiparallel linkage; for convenience, the two interacting RNA templates are presented in antiparallel orientation.