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. 2003 May;77(9):5065-72.
doi: 10.1128/jvi.77.9.5065-5072.2003.

Generation of novel syncytium-inducing and host range variants of ecotropic moloney murine leukemia virus in Mus spicilegus

Yong Tae Jung  1 , Christine A Kozak
Affiliations

Generation of novel syncytium-inducing and host range variants of ecotropic moloney murine leukemia virus in Mus spicilegus

Yong Tae Jung et al. J Virol. 2003 May.

Abstract

Mus spicilegus is an Eastern European wild mouse species that has previously been reported to harbor an unusual infectious ecotropic murine leukemia virus (MLV) and proviral envelope genes of a novel MLV subgroup. In the present study, M. spicilegus neonates were inoculated with Moloney ecotropic MLV (MoMLV). All 17 inoculated mice produced infectious ecotropic virus after 8 to 14 weeks, and two unusual phenotypes distinguished the isolates from MoMLV. First, most of the M. spicilegus isolates grew to equal titers on M. dunni and SC-1 cells, although MoMLV does not efficiently infect M. dunni cells. The deduced amino acid sequence of a representative clone differed from MoMLV by insertion of two serine residues within the VRA of SUenv. Modification of a molecular clone of MoMLV by the addition of these serines produced a virus that grows to high titer in M. dunni cells, establishing a role for these two serine residues in host range. A second unusual phenotype was found in only one of the M. spicilegus isolates, Spl574. Spl574 produces large syncytia of multinucleated giant cells in M. dunni cells, but its replication is restricted in other mouse cell lines. Sequencing and mutagenesis demonstrated that syncytium formation could be attributed to a single amino acid substitution within VRA, S82F. Thus, viruses with altered growth properties are selected during growth in M. spicilegus. The mutations associated with the host range and syncytium-inducing variants map to a key region of VRA known to govern interactions with the cell surface receptor, suggesting that the associated phenotypes may result from altered interactions with the unusual ecotropic virus mCAT1 receptor carried by M. dunni.

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Figures

FIG. 1.
FIG. 1.
(A) General structure of MoMLV. An open box is used to indicate the position of VRA. The arrows identify the PCR primers and their products. LTR, long terminal repeat. (B) The S82F substitution was introduced into MoMLV by a series of substitutions. The solid vertical lines indicate the positions of the three substitutions in the 2.5-kb env gene PCR product of Spl574 clone p480-10. The arrows indicate the restriction sites for BamHI (B) and HpaI (H). The internal HpaI fragment was first removed and replaced by the comparable segment of MoMLV to produce p480-Mo, and then the internal BamHI segment of p480-Mo was replaced by the S82F-containing BamHI segment of p480-10.
FIG. 2.
FIG. 2.
Comparison of the deduced amino acid sequences of the VRA regions of MoMLV and five other ecotropic MLVs. HoMLV, M. hortulanus (M. spicilegus) MLV (21, 22). MLV-SS is a Moloney clone into which two serine residues have been added. Dashes indicate no change; asterisks represent deletions.
FIG. 3.
FIG. 3.
(A) Uninfected M. dunni cells. (B) M. dunni cells 2 days after infection with Spl574. (C) Floating cells collected from an M. dunni culture 3 days after infection with Spl574. (D) M. dunni cells 2 days after infection with mutant virus MoMLV-S82F. (E) Uninfected NIH 3T3 cells. (F) NIH 3T3 cells 5 days after infection with Spl574. Objective lens magnification was ×10, except for panel C, for which magnification was ×40.
FIG. 4.
FIG. 4.
Deduced amino acid sequences of the env genes of three clones of the syncytium-inducing virus Spl574. The open boxes indicate the relative positions of the VRA, VRB, and PRD segments within SU. Vertical lines indicate the positions of substitutions or insertions in the three clones, and the numbers represent the amino acid position.
FIG. 5.
FIG. 5.
Comparison of the deduced amino acid sequences of the third extracellular regions of the mCAT1 receptor of NIH 3T3 (mCAT1), SC-1 (sCAT1), M. dunni (dCAT1), and M. spicilegus (HCAT1). The two potential N-linked glycosylation sites are underlined. Y235 and E237 are critical for efficient virus infection and binding of MLV envelope protein (1).
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