Figure 1.
(A) Schematic map of the 8176 nt genome of xmlv15. The LTR regions (R, U5, U3) are indicated with boxes. Two open reading frames encoding gag-pro-pol and env polyproteins are predicted. The corresponding start (AUG) and stop codons (UAA) are shown, along with their nucleotide positions. (B) Cloning and sequencing of the xmlvs. The clones obtained by PCR from SAM mouse genomic DNA (black bars) were sequenced. Primers used to amplify individual clones (Table 1) were derived from overlapping xmlv clones (arrows).
Table 1.
Oligodeoxyribonucleotide primer sequences used for cloning and RT-PCR.
Figure 2.
Phylogenetic analysis of the xmlvs.
Phylogenetic trees were constructed based on (A) the complete genome sequences of the xmlv15 and (B–C) the predicted gag, pro-pol and env polyproteins of xmlvs. The full length nucleotide sequences and the deduced amino acid sequences of the xmlvs as well as the corresponding sequences from identified MuLVs were aligned using ClustalX. The resulting alignments were used to generate neighbor-joining trees. The values at the branch nodes represent the percentage of confidence in a specific branching. The sequences of the xmlvs (xmlv15 and xmlv18) are highlighted in gray. The sequences used in the analysis and in the construction are as follows: non-ecotropic proviruses (mERVs), AKV MuLV, Friend MuLV, Moloney MuLV, Rauscher MuLV, Feline leukemia virus, Gibbon ape leukemia virus, Koala retrovirus, DG-75, MTCR, MuLV MCF 1233, XMRV VP35, MuLV NCI-417, MuLV NZB-9-1, XMRV 22Rv1, MX33 and Rmf2.
Figure 3.
Multiple sequence alignment of xmlvs.
(A) Sequence comparison of pol gene regions of xmlvs was performed with the corresponding regions of other X-MuLV isolates, DG-75 MuLV and XMRV VP62. (B) Multiple sequence alignment of the deduced amino acid sequences of xmlvs and related MuLVs spanning SU glycoprotein VRA, VRB and VRC, which were characterized by important regions for cellular tropism. This analysis employed the env protein sequences from the following viruses; AKV, Friend MuLV, Moloney MuLV, Rauscher MuLV, prototype polytropic clone MX27, DG-75, MTCR, MuLV MCF247, MuLV MCF 1233, MuLV NFS-Th-1 and MuLV NZB-9-1. The sequences were aligned using ClustalX. The dots indicate residues identical to those from xmlv15, and deleted residues appear as spaces.
Figure 4.
Detection of the xmlv proviruses.
(A) PCR results screening of the genomic DNA of SAM, C57BL and ICR mouse strains, hamsters and humans. (B) RT-PCR analysis of xmlvs in the brains of various mouse strains, including SAMP8, SAMR1, C57BL and ICR.