Figure 1.
A) Schematic representation of mA3. The nine exons are indicated by black boxes and the black arrows indicate the positions of the 7 PCR primers. The red arrow shows the position of the MLV LTR. B) PCR products of the indicated strains following amplification with primers a and b. C) The sequences of the 531 bp mA3 C57BL intron insertion and the LTR of the NZB-IU-6 X-MLV (GenBank No. K02730) were aligned using ClustalW [62] and marked to indicate the positions of the boundaries of the U3, R, and U5 regions and the CAT and TATA boxes. Asterisks indicate identical bases in the two sequences. D) Position of the inserted LTR in mA3. The insertion is present in C57BL and absent from BALB/c mice. A box encloses the splice donor site at the end of exon 2. The direct repeats flanking the LTR insertion are underlined and the LTR sequence is in red.
Figure 2.
Distribution of the X-MLV LTR in inbred strains and wild-derived M. musculus house mouse species.
An asterisk marks samples that were sequenced, and those examined for splice variants are underlined. Mice found to carry the virus restrictive C57BL mA3 sequence are bolded. CZECHI/CzI, CZECHII/CzII and LEWES/LW mice were sampled twice, from DNAs extracted from mice obtained from M. Potter in 1984, and from The Jackson Laboratory DNAs isolated from inbred lines developed from the same colonies. M. musculus mice trapped in California (CalWM) are listed without subspecies designation. Numbers in parentheses are the number of individuals tested. These 11 CalWM include mice trapped in Bouquet Canyon, Lake Casitas, and the SC-1 cell line.
Figure 3.
mA3 splicing patterns and expression levels in mouse strains and M. musculus subspecies.
A) RT-PCR products from the indicated sources using primers a and g. B) Splicing patterns observed in mRNAs of mice carrying LTR+ and LTR− mA3 alleles. Mice listed as having “other patterns” produce the exon5+ isoform with one or more smaller mRNAs. The dotted lines separate common laboratory strains and wild-derived mice. The tissue or cell source of RNA is indicated in {}. C) Quantitative real time PCR of mA3 transcripts in spleens of LTR+ and LTR− laboratory and wild-derived mouse strains. Amplification levels were normalized to β-actin. The mice producing the exon5+ isoform are indicated at the bottom. Sequence analysis of NFS/N and AKR mA3 indicates they carry the BALB/c allele.
Figure 4.
Positive selection on exons 2–4 of mA3 in Mus.
A) Cladogram showing branch values of dN/dS calculated using the free-ratio model of PAML, with the number of replacement and synonymous changes in parentheses. When dS = 0, dN/dS is infinite (Inf). dN/dS>1 suggests positive selection along that lineage. Bootstrap support for this tree topology was generally good, with most bootstrap percentages >90%. The thin lines represent branches with bootstrap values <70%. Colors indicate Mus subgenera: brown, Coelomys; green, Pyromys; blue, Nannomys; red, Mus with Mus house mouse (M. musculus) strains and species in pink. A purple arrow indicates insertion of the X-MLV LTR, and purple plus signs identify taxa carrying the LTR. B) Likelihood ratio tests were used to test for positive selection. Neutral models (M1, M7) were compared with selection models (M2, M8) using two different models of codon frequency (F3X4 or F61). P values <0.0001 provide strong evidence of positive selection. Tree 1 is the data-derived tree and tree 2 is the taxonomy-derived tree. Tree length is the average number of substitutions per codon along all branches. dN/dS ratio is given for the codons under selection, along with the % of codons in this category.
Table 1.
Sites that distinguish the mA3 genes in virus-restrictive C57BL and virus-nonrestrictive BALB/c mice, and sites under positive selection.
Figure 5.
mA3 sites under positive selection in the N-terminal Z2 CDA.
At the top is a diagram of the mA3 segment (exons 2–4) that encodes the N-terminal CDA showing the locations of the 9 codons that differ in virus restrictive C57BL and virus nonrestrictive BALB/c mice. At the bottom is a graph showing the posterior probability of positive selection at each codon based on an analysis of 26 mA3 sequences using codon frequency model F3X4 and selection Model 8 on the data-based trees (Table S2). A red line marks P = 0.95. The red arrows indicate the 7 codons that are strongly positively selected (P>0.99); these include 5 of the 9 polymorphic codons (long arrows) and codons 136 and 183. Codon 138 is positively selected at 0.99>P>0.95.
Figure 6.
Comparative sequence and structure of the active cytidine deaminase regions of hA3G and mA3.
A) Sequence alignment of the C57BL mA3 CDA (GenBank No. NM_030255) with that of hA3G (GenBank No. NM_021822), using CLUSTAL X [62]. Gray shading, identical residues. Red letters, AC LOOP 1. Blue letters, AC LOOP 3. Blue lines, α-helices. Blue arrows, β-strands. Blue dots, residues associated with the hA3G substrate groove that are important for deamination [32]–[34]. Green boxes, sites under positive selection in mA3. Red triangles, sites that are polymorphic between BALB/c and C57BL. B) Superimposed structures of hA3G-3IR2 and the mA3 homology model. α-helices are numbered. AC loops 1 and 3 are indicated. The red ball is zinc. C) Homology model of mA3 showing the 3 AC loop 1 residues under positive selection (yellow), and the 5 residues under strong (P>0.95) and very strong (P>0.99) selection that are on the opposite side of the substrate groove (red). D) Surface representation of mA3 showing the location of the predicted substrate groove (green line) and the two clusters of selected residues in blue and red.
Figure 7.
Antiviral activity of mA3 mutants carrying BALB/c residues at the 34–38 and 134–139 clusters.
293T cells were cotransfected with Friend virus clone pLRB302 and the indicated mA3 variants. The experiment was repeated six times with some experimental variations; shown are two representative experiments. One experiment (left panel) used 3 µg of pLRB302 and 1 µg of mA3. The second experiment (right panel) used 4 µg of pLRB302 and 0.5 µg of mA3, and total DNA amounts were adjusted to 6 µg with empty pcDNA3.1(−) vector DNA; mA3 was reduced because titration experiments showed 0.5 µg to have antiviral activity (not shown). Infectivity of collected Friend virus was assayed by the XC overlay test normalized against reverse transcriptase activity (experiment 1) or virus capsid protein in pelleted virus (experiment 2). Infectivity of virus produced in the absence of mA3 was defined as 100%, and infectivity of the other viruses is given as a percentage of the control. Virus infectivity was determined 6 times in both experiments.