Cell, Vol. 14.959-970, August 1978. Copyright 0 1978 by MIT Heteroduplex Analysis of the Nonhomology Region between Moloney MuLV and the Dual Host Range Derivative HIX Virus Daniel J. Donoghue, Ellen Rothenberg,* Nancy Hopkins, David Baltimore and Phillip A. Sharp Department of Biology and Center for Cancer Research Massachusetts Institute of Technology Cambridge, Massachusetts 02139 * Memorial Sloan-Kettering Cancer Center New York, New York 10021 Summary The dual host range virus HIX has been previ- ously characterized as an envelope gene recom- binant between Moloney murine leukemia virus (MO-MuLV) and an unidentified xenotropic murine leukemia virus. Using long reverse transcripts of MO-MuLV, a region of nonhomology has been mapped by electron microscopic analysis of het- eroduplexes formed with HIX 3% virion RNA. In this nonhomology region, the MO-MuLV cDNA strand measured approximately 900 nucleotides, mapping between 1.6 and 2.5 kilobases from the 3’ end. In a previous study, hybridization of MO-MuLV 21s RNA with MO-MuLV cDNA resulted in the formation of different heteroduplex structures di- agnostic of a noncontiguously coded leader se- quence at the 5’ end of the 21s RNA. Following hybridization of poly(A)+ HIX 21s RNA with 8.2 kb MO-MuLV cDNA, analogous heteroduplex structures were observed exhibiting the Mo- MuLV:HIX substitution loop in the DNA:RNA seg- ment of the molecules. This analysis permitted more precise mapping of the nonhomology region with respect to the splice point in the 21s pre- sumptive glycoprotein mRNA. The mapping of this nonhomology region in HIX virus provides an internal visual marker for the 3’ end of the ge- nome which may prove useful in future analyses of other deletion or substitution derivatives of Mo- MuLV. Introduction Murine leukemia viruses (MuLVs) can be classified according to their biological host range into the ecotropic viruses, those which replicate in mouse cells but are restricted in cells of other species, and the xenotropic viruses (MuXs), those which replicate in cells of many nonmurine species but are restricted in mouse cells (Levy, 1973; Aaronson and Stephenson, 1973; Benveniste, Lieber and Todaro, 1974). In addition, amphotropic viruses have also been isolated whose host range includes both mouse cells and cells of other species (Hartley and Rowe, 1976; Rasheed, Gardner and Chan, 1976). By a number of criteria, the molecular spec- ificities responsible for these differences in host range appear to reside on the MuLV glycoprotein, designated gp70 and encoded by the envelope (env) gene (Fischinger et al., 1975b; DeLarco and Todaro, 1976; Besmer and Baltimore, 1977). A new class of murine retroviruses has recently been identified as distinct from the ecotropic, xen- otropic and amphotropic MuLVs; these constitute the dual host range viruses which apparently arise by recombination within the env gene between ecotropic and xenotropic MuLV parents (Fischin- ger, Nomura and Bolognesi, 1975a; Elder et al., 1977; Fischinger et al., 1977; Hartley et al., 1977; Failer, Rommelaere and Hopkins, 1978; Faller and Hopkins, 1978). The dual host range viruses have properties in common with both ecotropic and xenotropic MuLVs, as judged by interference and neutralization tests. Thus although the dual host range viruses have a host range similar to the amphotropic viruses, they are clearly distinguish- able on the basis of these other criteria. One such virus, named HIX virus (for hybrid properties of MuLV-IC and MUX), was identified after passage of the IC isolate of Moloney MuLV (MO-MuLV) through mouse cells (Fischinger et al., 1975a). HIX virus presumably represents the product of a recombi- nation event between MO-MuLV and a still uniden- tified strain of MUX. In an analogous fashion, the MCF viruses (for mink cell focus-inducing) have been isolated from leukemic and late preleukemic AKR mice, and presumably arise by recombination between AKR ecotropic virus and an unidentified xenotropic parent (Hartley et al., 1977). Evidence for the recombinational origin of dual host range viruses comes from biochemical studies of their genomes and their env gene products. Tryptic peptide analyses of the glycoproteins of both HIX and the MCF viruses suggest that the dual host range viruses have tryptic peptides in common with xenotropic gp7Os as well as with ecotropic gp7Os (Elder et al., 1977; J. Elder and P. Fischin- ger, personal communication). RNAase Tl oligo- nucleotide mapping of HIX and MCF virion RNAs similarly indicates that the dual host range viruses have lost ecotropic-specific oligonucleotides from the 3’ portion of the genome; these have been replaced with HIX- or MCF-specific oligonucleo- tides presumably derived from the xenotropic par- ents (Faller et al., 1978; Rommelaere et al., 1978). It is assumed that at least a fraction of these altered oligonucleotides corresponds to sequences en- coding the altered glycoproteins. The 35s virion RNA of murine retroviruses is translated to yield the internal structure (gag) proteins and reverse transcriptase @o/), whereas the env glycoprotein appears to be translated from