Virological properties and nucleotide sequences of Cas-E-type endogenous ecotropic murine leukemia viruses in South Asian wild mice, Mus musculus castaneus

Abstract

Two types of endogenous ecotropic murine leukemia viruses (MuLVs), termed AKV- and Cas-E-type MuLVs, differ in nucleotide sequence and distribution in wild mouse subspecies. In contrast to AKV-type MuLV, Cas-E-type MuLV is not carried by common laboratory mice. Wild mice of Mus musculus (M. m.) castaneus carry multiple copies of Cas-E-type endogenous MuLV, including the Fv-4(r) gene that is a truncated form of integrated MuLV and functions as a host's resistance gene against ecotropic MuLV infection. Our genetic cross experiments showed that only the Fv-4(r) gene was associated with resistance to ecotropic F-MuLV infection. Because the spontaneous expression of infectious virus was not detected in M. m. castaneus, we generated mice that did not carry the Fv-4(r) gene but did carry a single or a few endogenous MuLV loci. In mice not carrying the Fv-4(r) gene, infectious MuLVs were isolated in association with three of six Cas-E-type endogenous MuLV loci. The isolated viruses showed a weak syncytium-forming activity for XC cells, an interfering property of ecotropic MuLV, and a slight antigenic variation. Two genomic DNAs containing endogenous Cas-E-type MuLV were cloned and partially sequenced. All of the Cas-E-type endogenous MuLVs were closely related, hybrid-type viruses with an ecotropic env gene and a xenotropic long terminal repeat. Duplications and a deletion were found in a restricted region of the hypervariable proline-rich region of Env glycoprotein.

FIG. 1

Southern blot analysis of Cas-E-type endogenous MuLVs. HindIII-digested mouse DNAs were hybridized with the Fv-4^renv probe (19). (A) Lane 1, (WHT × Tgt)F₁; lane 2, Bgr. (B) Lane 1, Bgr; lane 2, N.Fv-4^r (= Frg2); lane 3, N.Frg3; lane 4, N.Frg4; lane 5, N.Frg6; lane 6, N.Frg1 and -3; lane 7, N.Frg6 and -7. Mice of lanes 2 to 7, which were produced by backcrossing a Bgr mouse to NFS/N mice, carried the indicated endogenous MuLV(s) derived from the Bgr mouse. The Frg5 band was detectable in two Bgr mice (panel A and data not shown) but not in another Bgr mouse (panel B). The Frg5 band did not show a Mendelian inheritance (see text).

FIG. 2

Segregation of splenomegaly and F-MuLV replication in NFS × (NFS × Bgr)F₁ (left panel) and NFS × (WHT × Ttg)F₁ (right panel) backcross mice after injection of Friend leukemia virus complex. The closed circles and the open circles indicate mice carrying Fv-4^rs or not carrying the Fv-4^r gene (Fv-4^ss), respectively. Ten days after virus injection, the spleen weight of an individual mouse was measured, and the spleen homogenates were used for titration of F-MuLV by the UV-XC test on C-182 cells. Liver or tail DNA of each mouse was used for Southern blot analysis, as shown in Fig. 1. An arrow attached to the circle means that the virus was undetectable and, thereby, was below the indicated titer.

FIG. 3

Membrane immunofluorescence analysis of cells infected by Frg3 virus. SC-1, YH-7, D8b5, and D3h1g cells were infected by an undiluted or a 10-fold diluted solution of an Frg3 virus stock. Five days after the infection, the cells were treated with BALB/c anti-C4W serum followed by fluorescent isothiocyanate-conjugated anti-mouse IgG. Immunofluorescence (IF) intensities were measured by a flow cytometer, and the results are shown in histograms.

FIG. 4

Interfering activity of the Frg3, Frg4, and Frg6 viruses toward superinfection. SC-1 cells persistently infected with the viruses were superinfected with MSV pseudotyped with amphotropic (4070A), dualtropic (AKR13), or ecotropic (AKV623, Friend, and Moloney) MuLVs. MSV-induced foci were counted 5 to 7 days after infection.

FIG. 5

Structure of Frg1 and Frg3 endogenous MuLVs. Compared to a wild-type endogenous MuLV, Frg1 MuLV had a deletion from gag to env genes and Frg3 had complete env and 3′ LTR regions. The nucleotide sequences have been deposited in DDBJ under accession no. AB050720 (Frg1) and no. AB050721 (Frg3).

FIG. 6

Phylogenetic analyses of env and LTR U3 genes of Cas-E-type MuLVs. (A) Deduced amino acid sequences of the env genes were analyzed by the unweighted pair-group method with arithmetic means (UPGMA). Sequence sources were as follows: Friend (31), Moloney (53), AKV (17), Fv-4 (19, 41), Cas-Br-E (46), hortulanus eco (59), modified polytropic Mx33 (55), polytropic Mx27 (55), NZB xeno (43), Moloney MCF (4), and amphotropic 4070A (45). (B) The U3 sequences were aligned according to the characteristic sequences (28, 58) (see Fig. 8), and then the phylogenetic analysis was done by the UPGMA method in combination with Kimura's two-parameter method. Sequence sources were as follows: Mxv2 (58), Mxv7 (58), Mxv28 (58), Mcv3 (58), Mcv11 (58), Mxv11 (58), Mcv18 (58), NFS-Th-1 (28), NZB xeno (43), and Fv-4 (19).

FIG. 7

Alignment of the amino acid sequences of the four Cas-E-type MuLV env genes. Regions I, II, and III represent the disulfide-bonded structural elements conserved in ecotropic MuLVs (38). The hypervariable proline-rich region is indicated. The 8-aa direct repeats found in the proline-rich region of Frg1 and Fv-4^r are underlined with a dotted line and a double line, respectively. A 5-aa deletion of Cas-Br-E, compared with Frg3, is boxed. Asterisks or dots shown under the sequences indicate the position in which all of the four MuLVs have the same amino acid or in which three of the four MuLVs have the same amino acid, respectively.

FIG. 8

Alignment of LTRU3 sequences of Cas-E-type ecotropic MuLVs and xenotropic MuLVs. Based on the characteristic sequences (1, 1∗, 2, 4, 4∗, 5, 5∗, 6, and 6∗) (28, 58), the U3 sequences of Cas-E-type MuLV (Fv-4, Frg3, and Frg1) were aligned, along with the xenotropic U3 sequences. Dots indicate the nucleotide identity and dashes indicate the absence of a nucleotide. Xenotropic U3 sequences are grouped into X-1 (NFS-Th-1, NZB xeno, and Mcv18), X-II (Mxv2 and Mxv7), X-III (Mcv11 and Mcv3), and X-IV (Mxv28 and Mxv11) (58).