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. 2005 Jul;79(13):8316-29.
doi: 10.1128/JVI.79.13.8316-8329.2005.

Characterization of hortulanus endogenous murine leukemia virus, an endogenous provirus that encodes an infectious murine leukemia virus of a novel subgroup

Christopher H Tipper  1 , Craig E Bencsics, John M Coffin
Affiliations

Characterization of hortulanus endogenous murine leukemia virus, an endogenous provirus that encodes an infectious murine leukemia virus of a novel subgroup

Christopher H Tipper et al. J Virol. 2005 Jul.

Abstract

Simple retroviruses present a unique opportunity for examining the host-virus relationship. Following exogenous infection and integration into the germ line, copies of these viruses can become fixed within the genome. The resulting endogenous proviral "fossils" represent a record of past retroviral infections and forms. Previous work in our laboratory has been directed at dissecting the extensive nonecotropic murine leukemia virus content of the mouse genome. One such provirus, hortulanus endogenous murine leukemia virus (HEMV), found in a single copy in the genome of Mus spicilegus, was remarkable for characteristics that suggested that it was ancient and related to the hypothetical common ancestor of murine leukemia viruses (MLVs) and other gammaretroviral species. In the present study, we have analyzed its functional properties. Transfection of a molecular clone of the HEMV provirus into mouse-derived cell lines revealed that it is replication competent. Furthermore, host range and interference studies revealed a strictly ecotropic host range and the use of a receptor distinct from those used by other classical MLVs. The identity of nucleotide sequence of the long terminal repeats (LTRs) further suggested that HEMV is a relatively recent insertion into the M. spicilegus genome at the distal end of chromosome 7. Although unique to M. spicilegus, its presence in a homozygous state in three individuals obtained from different regions implies that it has been present long enough to become fixed in this species. Exhaustive phylogenetic analysis of all regions of the HEMV genome supported the previously assigned ancestral position of HEMV relative to other MLV-related viruses. Thus, HEMV is a relatively recent introduction into the Mus germ line but is representative of a relatively ancestral MLV group.

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Figures

FIG. 1.
FIG. 1.
Pseudotype construction and LacZ+ PuroR virus production. (A) The HEMV pseudotype was constructed by PCR amplification and cloning of a region including the env gene from M. spicilegus DNA. The amplified fragment was swapped into the ecotropic packaging-deficient vector pSV-Ψ minus-E-MLV. This construct, pSV-Ψ minus-HEMV-MLV, and the packagable marker vector pLacPuro were transfected into 293T cells. Viral particles produced by these cells were then used to infect tissue culture cells. Infection efficiency was assessed by staining for β-galactosidase. (B) A similar method was used to make other LacZ+ PuroR pseudotypes. A triple transfection method using pMLVgagpol, pLacPuro, and various env gene constructs produced these viruses.
FIG. 2.
FIG. 2.
Construction of the complete HEMV proviral vector pHEMV-TOPO. HEMV was originally cloned and sequenced piecemeal following the successful amplification of the fragments shown from the M. spicilegus genome. The final construct was derived primarily from HEMV6 coupled with the 5′ LTR and 3′ LTR regions. The final construct was ligated into the vector pCR2.1-TOPO. Amplicons acquired and used for initial analysis and sequencing are colored light grey. Amplicons acquired and utilized, or partially utilized, in the final construction of the vector are colored black. The final vector construct was made by ligating together the indicated sequences cut with the indicated restriction endonucleases. Primers used to acquire each amplicon are shown associated with each sequence.
FIG. 3.
FIG. 3.
Comparison of the HEMV and MLV genomes. The HEMV genome is shown aligned with that of MoMLV (39). The two genomes differ by 232 bases, most of which are in two regions: the foreshortened VRA region in SU and the enhancer region of U3 (46). Two other differences in the otherwise-well-aligned genomes are worth noting: the HEMV MA protein is 6 amino acids shorter than Moloney MLV MA, and there are insertions of 4 amino acids in the C terminus of the HEMV IN and 1 amino acid in HEMV p12. The IN insertion and 4 of the 6 residues lacking in MA are, at this time, unique to HEMV.
FIG. 4.
FIG. 4.
The complete HEMV clone can yield virus infectious for NIH 3T3 cells. (A) NIH 3T3 cells were transfected with complete proviral constructs of HEMV, Moloney MLV, and 10A1. Virus production and spread were followed by reverse transcriptase activity in the culture medium, as determined by the amount of incorporated [32P]TTP into poly(dT) using a poly(A)-oligo(dT) primer-template. Samples were collected every 3 days, prior to passaging the cells, and were counted in triplicate. The samples that contained the most RT activity (passage 4 for Moloney MLV and passage 5 for HEMV and 10A1) were filtered and used to infect the other mouse cell lines, SC1 (B) and MMK (C). Mock transfections were treated in exactly the same manner except that no viral DNA was included in the transfection procedure.
FIG. 5.
FIG. 5.
Alignment of the HEMV integration junctions with mouse chromosome 7. The HEMV junction fragments were cloned by ligation-mediated PCR and sequenced. The integration site was located in the mouse genome sequence database (http://www.ensembl.org/Mus_musculus/). Data from both the 5′ and 3′ flanking sequences are in agreement that HEMV is integrated into what should be the distal end of M. spicilegus chromosome 7. The genomes of M. spicilegus, M. macedonicus, M cookii, and M castaneus are all slightly polymorphic within 50 bp of the integration site with respect to the reference C57BL/6 sequence. M. spicilegus sequence was confirmed by sequencing at least two independent clones for each junction in both directions. Highlighted are the four host residues that flank the integration site within M. spicilegus aligned with the unoccupied sites within the other genomes as well as the TG and CA dinucleotides that mark the termini of the provirus.
FIG. 6.
FIG. 6.
Occupation of the HEMV integration site. (A) Schematic of mouse chromosome 7, complete with HEMV provirus integrated at a position corresponding to C57BL/6 residue 125,863,352 (of 137,389,636) and including the locations of primers used in the PCR amplification experiment in panels B, C, D, and E. (B) Amplification with the primer pair 3LTRF and Int6R should detect a 1,499-bp junction fragment only if the HEMV provirus is present at the same site. (C) Amplification with the primer pair Int5F and Int6R was used to detect the 1,539 bp predicted for the unoccupied integration site. The results for M. caroli, M. dunni, M. (coelomus) pahari, and M. (pyromus) platythrix are discussed in the text. The presence of an HEMV-like provirus was detected by PCR amplification with HEMV VRA (D)- and LTR (E)-specific primers only in the three M. spicilegus individuals and the tail fibroblast DNA from M. spicilegus (Pancevo) used in the tropism experiments. φX174/HaeIII markers were run in lane 1, and lambda/HindIII markers were run in lane 20. Lanes: a, AKR/J; b, M. m. domesticus (Zalende); c, M. m. musculus (CzechII), d, M. m. molossinus (MolC); e, M. castaneus (Cast/Ei); f, M. bactrianus (BIR); g, M. spretus (Spret/Ei); h, M. macedonicus (XBS); i, M. spicilegus (Pancevo); j, M. spicilegus (Halbturn); k, M. spicilegus (ZRU); l, M. cervicolor (popaeus); m, M. cervicolor (cervicolor); n, M. caroli; o, M. cookii; p, M. dunni; q, M. (coelomus) pahari; r, M. (pyromus) platythrix; s, M. spicilegus (Pancevo) (tail fibroblast preparation).
FIG. 7.
FIG. 7.
Phylogenetic analysis of regions of MLV-related genomes. DNA sequences of the viruses shown were analyzed using maximum parsimony with the exhaustive search option (42). (A) Reverse transcriptase. (B) The TM region of the Env protein. (C) The SU region. (D) The gag region. Boxes in A, B, and D indicate endogenous and exogenous viruses; boxes in C indicate different subgroups. The sequence of the exogenous xenotropic virus DG-75 was used in this analysis, as it is the only complete virus of this subgroup available (37). It should be noted that it is highly possible that despite its distinctly xenotropic env region, this virus is a recombinant between an endogenous xenotropic locus and some other MLV. Regardless, initial phylogenetic analyses that included other established partial xenotropic sequences did not distinguish DG-75, so we consider it to be an example of this endogenous subgroup.
FIG. 8.
FIG. 8.
Ten million years of mouse evolution. The cladogram shows recognized mouse taxa and their relationship as inferred by several molecular techniques. M. m. molossinus, endemic to Japan, is a hybrid of M. m. musculus and M. m. castaneus. The myriad strains of laboratory mouse are derived primarily from the fancy mouse breeding fashionable in Europe (M. m. domesticus) and Japan (M. m. molossinus) in the 19th century. The lighter grey box indicates species derived from a common ancestor before the creation of the West Palearctic species M. spretus, M. macedonicus, and M. spicilegus. Endogenous MLVs have been confirmed to reside only in these species. The darker grey box highlights the HEMV host M. spicilegus (this figure is a composite work from references , , and 35).
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