Molecular and phylogenetic analyses of a new amphotropic murine leukemia virus (MuLV-1313)

Abstract

Background: The amphotropic murine leukemia viruses (MuLV-A's) are naturally occurring, exogenously acquired gammaretroviruses that are indigenous to the Southern California wild mice. These viruses replicate in a wide range of cell types including human cells in vitro and they can cause both hematological and neurological disorders in feral as well as in the inbred laboratory mice. Since MuLV-A's also exhibit discrete interference and neutralization properties, the envelope proteins of these viruses have been extremely useful for studying virus-host cell interactions and as vehicles for transfer of foreign genes into a variety of hosts including human cells. However, the genomic structure of any of the several known MuLV-A's has not been established and the evolutionary relationship of amphotropic retroviruses to the numerous exogenous or endogenous MuLV strains remains elusive. Herein we present a complete genetic structure of a novel amphotropic virus designated MuLV-1313 and demonstrate that this retrovirus together with other MuLV-A's belongs to a distinct molecular, biological and phylogenetic class among the MuLV strains isolated from a large number of the laboratory inbred or feral mice.

Results: The host range of MuLV-1313 is similar to the previously isolated MuLV-A's except that this virus replicates efficiently in mammalian as well as in chicken cells. Compared to ENV proteins of other MuLV-A's (4070A, 1504A and 10A-1), the gp70 protein of MuLV-1313 exhibits differences in its signal peptides and the proline-rich hinge regions. However, the MuLV-1313 envelope protein is totally unrelated to those present in a broad range of murine retroviruses that have been isolated from various inbred and feral mice globally. Genetic analysis of the entire MuLV-1313 genome by dot plot analyses, which compares each nucleotide of one genome with the corresponding nucleotide of another, revealed that the genome of this virus, with the exception of the env gene, is more closely related to the biologically distinct wild mouse ecotropic retrovirus (Cas-Br-E) isolated from another region of the Southern California, than to any of the 15 MuLV strains whose full-length sequences are present in the GenBank. This finding was corroborated by phylogenetic analyses and hierarchical clustering of the entire genomic sequence of MuLV-1313, which also placed all MULV-A's in a genetically distinct category among the large family of retroviruses isolated from numerous mouse strains globally. Likewise, construction of separate dendrograms for each of the Gag, Pol and Env proteins of MuLV-1313 demonstrated that the amphotropic retroviruses belong to a phylogenetically exclusive group of gammaretroviruses compared to all known MuLV strains.

Conclusion: The molecular, biological and phylogenetic properties of amphotropic retroviruses including MuLV-1313 are distinct compared to a large family of exogenously- or endogenously-transmitted ecotropic, polytropic and xenotropic MuLV strains of the laboratory and feral mice. Further, both the naturally occurring amphotropic and a biologically discrete ecotropic retrovirus of the Southern California wild mice are more closely related to each other on the evolutionary tree than any other mammalian gammaretrovirus indicating a common origin of these viruses. This is the first report of a complete genomic analysis of a unique group of phylogenetically distinct amphotropic virus.

Figure 1

Chicken and rat embryo cell cultures infected with MuLV-1313 pseudotypes of RSV/src genome. Panel A- Uninfected chicken embryo C/O cells ; Panel B- a culture of same chicken cells as in A, infected with MuLV-1313 pseudotypes of the Rous Sacoma virus (RSV) associated src genome ; Panel C- uninfected Fischer rat embryo fibroblasts and Panel D- same rat embryo cells infected with MuLV-1313 pseudotypes of RSV/src genome. Note the classical rounded cell morphology of the RSV- transformed cells. The virus infected cultures produced excess helper virus (MuLV-1313) as detected by the presence of reverse transcriptase activity in the culture fluid.

Figure 2

Relationship of MuLV 1313 gPr80 Env protein with those of other amphotropic MuLV's. All deduced amino acid sequences of the MuLV 1313 gPr80 Env proteins and related amphotropic MuLV strains 4070A, and 10A-I were aligned using progressive, pair-wise alignments implemented in the Pileup and the Gap programs of the Wisconsin Package (version 9.0), Genetics Computer Group (GCG), Madison, WI [63-67, 80]. The amino acid sequences of MuLV 1313 Env protein are shown in its entirety (represented in bold, capital letters using the standard single-letter symbols). Amino acid positions that are in total agreement with MuLV 1313 Env are indicated with a dash (-) and differences are represented as small letters. Periods (·) in the 10A-I Env protein sequence indicate spaces that were introduced to maximize the alignment. Boundaries of the Env signal peptide (Leader) and the mature processed proteins are labeled and indicated with bold vertical lines. Major landmarks of the extracellular gp70 surface (SU) protein include the (i) variable region A (VRA), (ii) VRB and (iii) proline-rich region. Major landmarks of the p15E transmembrane™ protein include the (i) fusion, (ii) heptad repeat, (iii) membrane spanning and (iv) the R-peptide or p2E domains. Eight potential N-linked glycosylation sites (N, X, S/T) in the SU proteins of the MuLV 1313 and 4070A are underlined. Disulfide linkages shown at the N-terminus of SU are based on those deduced for the polytropic envelope protein [81]. The boundaries of the various elements included in this diagram are based on previously published work [51, 82, 83]. Accession numbers for each of the env genes used in this analysis are MuLV 1313 (AF411814), M33469 for the naturally occurring amphotropic virus 4070 and M33470 for the recombinant amphotropic virus 10A-1.

Figure 3

Dot Martrix analyses of the whole MuLV-1313 genome. Dot plots of the MuLV-1313 genome (GenBank accession number AF411814) were constructed utilizing the COMPARE which produced files of 15621 points of full-length MuLV genomes. Dot matrix was constructed using DOTPLOT programs of the Wisconsin Package, Version 9.0, Genetics Computer Group (GCG), Madison WI and Vector NTI (Invitrogen, Carlsbad, California) tool with windows setting at 21 and stringency at 44. This analysis compares each nucleotide position with the corresponding position of another genome (Dot). Solid diagonal line represents similarity and broken lines indicate gaps. Although Dot-Matrix analyses were performed on several MuLV strains, viruses that showed high similarity scores are shown in panels A, B and C (see Additional File 2). Dot Matrix analyses of full-length genomic sequences shown include ; Panel A, Cas-Br-E [25] (X57540); Panel B, AKV (J01998), and Panel C, Moloney (J02255). The highest nucleotide similarity is observed with the CAS-Br-E ecotropic virus isolated from a Southern California Wild mouse with paralysis (Panel A). This is followed by Moloney [8] and AKV MuLV strains [85] (Panels B & C respectively). Note, the env sequences of MuLV-1313 are totally unrelated to all three viruses shown by large gap in this area of the diagonal line. In addition, note the numerous broken lines in gag and pol regions of the Moloney and AKV MuLV genomes.

Figure 4

Phylogenetic analyses of full Length MuLV genomes. Full-length nucleotide sequences as well as deduced amino acid sequences of 15 MuLV strains including MuLV-1313 were aligned and all gaps were stripped from the alignments before the phylogenetic trees were constructed and bootstrapping was set at 1000. Phylogenetic analyses were performed using PHYLIP [66, 71]. PHYLIP packages SEQBOOT, PROTDIST, DNADIST, NEIGHBOR, CONSENSE, and DRAWGRAM. The original data set was first analyzed by SEQBOOT which produced 100 bootstrapped data sets. The distance matrices on these data sets were achieved using PROTDIST for amino acid sequences and DNADIST for nucleotide sequences. The distance matrices were joined using NEIGHBOR. The tree files from NEIGHBOR were then applied with CONSENSE and the consensus tree was drawn using DRAWGRAM. Multiple sequence alignment were made using Vector NTI (Invitrogen, Carlsbad, California) with default gap opening penalty of 15 and default gap extension penalty of 6.66. Full length genomes used in the construction of the dendrogram included; AKV MuLV (J01998), MuLV 1313 (AF411814), Cas-Br-E MuLV (X57540), Friend-57 MuLV (X02794), Friend FB29 MuLV (Z11128), Friend PVC211 MuLV (M93134), Friend (FrC6-A8F5 D88386), mink cell focus-forming virus 1233 (MCF1233, U13766), Moloney MuLV (J02255), radiation leukemia virus (RadLV, K03363), Rauscher MuLV (Rauscher, U94692), SL3-3 MuLV (AF169256), solid-type reticulum cell sarcoma 19-6 MuLV (SRS 19-6, AF019230), HEMV (AY818896) and MDEV (AF053745). Note that both the amphotropic MuLV-1313 and ecotropic Cas-Br-E MuLV of the Southern California feral mice arise from a separate node of the phylogenetic tree indicating their evolutionary relationship.

Figure 5

The phylogenetic tree of MuLV gag genes. Dendrogram was constructed using deduced amino acid sequences of the entire gag gene of MuLV-1313 and 14 other full-length Gag sequences available in the databases. Note the evolutionary relationship of the highly conserved gag genes of the ecotropic Cas-Br-E and amphotropic MuLV-1313 viruses isolated from the wild mice of the Southern California regardless of their differences in their host range. Sequences used in the analysis include: AKV MuLV (J01998), MuLV- 1313 (AF411814), Cas-Br-E MuLV (X57540), Friend-57 MuLV (X02794), Friend FB29 MuLV (Z11128), Friend PVC211 MuLV (M93134), Friend (FrC6-A8F5 D88386), mink cell focus-forming virus 1233 (MCF1233, U13766), Moloney MuLV (J02255), radiation leukemia virus (RadLV, K03363), Rauscher MuLV (Rauscher, U94692), SL3-3 MuLV (AF169256) and solid-type reticulum cell sarcoma 19-6 MuLV (SRS 19-6, AF019230), HEMV (AY818896) and MDEV (AF053745). Note the segregation of MuLV- 1313 and Cas-Br-E MuLV on a distinct node of the phylogenetic tree.

Figure 6

Evolutionary relationship between highly conserved MuLV pol genes. Dendrogram was constructed from a 576 bp region of the MuLV pol gene encoding the highly conserved domains 1 through 7 of the retroviral reverse transcriptase protein [67] by using the neighbor-joining method. No gaps were introduced in any of the respective sequences to maintain the final alignment. Horizontal branch lengths are drawn to scale. Sequences from 13 highly conserved regions of 13 viruses used in the analysis include: MuLV 1313 (AF411814), Cas-Br-E MuLV (X57540), AKV MuLV (J01998), Friend-57 MuLV (X02794), Friend FB29 MuLV (Z11128), Friend PVC211 MuLV (M93134), mink cell focus-forming virus 1233 (MCF1233, U13766), Moloney MuLV (J02255), radiation leukemia virus (RadLV, K03363), Rauscher MuLV (Rauscher, U94692), SL3-3 MuLV (AF169256) and solid-type reticulum cell sarcoma 19-6 MuLV (SRS 19-6, AF019230) and HEMV (AY818896). Comparison of highly conserved pol sequences also indicated that MuLV 1313 is distinct from all other MuLV strains and is related to Cas-Br-E MuLV.

Figure 7

Phylogenetic analyses of amino acid sequences of complete MuLV env genes. The env genes of 26 MuLV strains were compared with those of MuLV 1313(AF411814) and the evolutionary tree was constructed using deduced amino acid sequences of full-length MuLV env genes and gaps were stripped from the alignment prior to construction of the final tree. Viruses included are: Friend FrC6-A8F5 MuLV (D88386); Friend PVC-211 MuLV (M93134); Rauscher MuLV (U94692); Friend SFFV-spleen focus-forming virus (AF030173); Friend MuLV (X02794); SRS 19-6 MuLV (AF019230) [82]; Moloney MuLV (J02255) [85]; AKV MuLV(J01998) ; Cas-Br-E MuLV (X57540) ; HoMuLV-Mus hortalanus murine leukemia virus (M26527) ; 4070A MuLV (M33469) ; 1313 MuLV(AF411814); 10A1 MuLV (M33470); NZB-9-1 MuLV (K02730); CWM-S-5X MuLV (M59793); HRS/J MuLV (M17326); Friend 98 MCF-mink cell focus-forming virus (AF133256); Friend MCF (X01679); Rauscher MCF (M10100); Moloney MCF (J02254); CI-3 MCF (K02725), 1233 MCF (U13766), MDEV (AF053745), HEMV (AY818896), M813 (AF327437) RadLV (K03363) and SL3-3 (AF16925). Note the unique separation of amphotropic viruses on a single node with a distinct branch for MuLV-1313.

Figure 8

Phylogenetic relationship of MuLV 1313 to various mammalian gammaretroviruses. This analysis utilized full-length or nearly full-length Env proteins 29 retroviruses isolated from mouse, rat, cat, pig, monkey and gibbon ape. The original multiple sequence alignment was created with Clustal W and the rooted tree phenogram was constructed using pre-aligned sequences and PHYLIP [64-66, 71, 80]. Abbreviations and accession numbers (in brackets) of viruses used in the phenogram are according to the GenBank database SSAV-simian sarcoma-associated virus (AF055064); GALV-gibbon ape leukemia virus (M26927); PERV-porcine endogenous virus (Y17013); MDEV-Mus dunni endogenous virus (AF053745); RaLV-rat leukemia virus (M77194), FeLV A-feline leukemia virus, subgroup A (M18247); FeLV B-feline leukemia virus, subgroup B (Gardner-Arnstein strain) (X00188); MuLV-murine leukemia virus MuLV 1313 (AF411814); Friend FrC6-A8F5 MuLV (D88386); Friend PVC-211 MuLV (M93134); Rauscher MuLV (U94692); Friend SFFV-spleen focus-forming virus (AF030173); Friend MuLV (X02794); SRS 19-6 MuLV (AF019230) [83]; Moloney MuLV (J02255); AKV MuLV(J01998) ; Cas-Br-E MuLV (X57540) ; HoMuLV-Mus hortalanus murine leukemia virus (M26527) ; 4070A MuLV (M33469) ; 10A1 MuLV (M33470); NZB-9-1 MuLV (K02730); CWM-S-5X MuLV (M59793); HRS/J MuLV (M17326); Friend 98 MCF-mink cell focus-forming virus (AF133256); Friend MCF (X01679); Moloney MCF (J02254); CI-3 MCF (K02725), 1233 MCF (U13766), MDEV (AF053745), RadLV (K03363) and SL3-3 (AF16925)

Figure 9

Similarity Plot of Full-length MuLV genomes: Plots of similarity between the MuLV 1313 genome (the 'query' sequence) and 10 representative, full-length MuLV genomes (the 'reference' sequences) present in the GenBank database were generated by SimPlot [72]. This program plots similarity versus position and calculates percent identity of a query sequence to a panel of reference sequences after their alignment in a sliding window, which is moved across the alignment in steps. The window and step sizes are adjustable. Prior to this analysis, an alignment of the full-length MuLV genomes was generated by the Clustal W program [64, 80]. Each curve is a comparison between the query genome being analyzed and one of the reference genomes after an alignment of the genomes has been made and positions containing gaps within the alignment were removed. Sequences used in the analysis include: AKV MuLV (AKV, J01998), Cas-Br-E MuLV (CAS-BR-E, X57540), Friend-57 MuLV (FR-MuLV 57, X02794), Friend FB29 MuLV (FR-MuLV FB29, Z11128), Friend PVC211 MuLV (FR-MuLV PVC211, M93134), mink cell focus-forming virus 1233 (MCF1233, U13766), Moloney MuLV (Moloney, J02255), Rauscher MuLV (Rauscher, U94692), SL3-3 MuLV (SL3-3, AF169256) and solid-type reticulum cell sarcoma 19-6 MuLV (SRS 19-6, AF019230). All 'reference' sequences used in the analysis with the exception of the polytropic MCF1233 display ecotropic in-vitro host range. Above the curves is a schematic diagram (drawn to scale) showing the relative positions of the coding regions of the gag (MA, pp12, CA and CA), pol (PR, RT and IN) and env (SU and TM) genes of the MuLV genome.