Retraction and Corrections

RETRACTION CORRECTIONS

PROFILE EVOLUTION Retraction for ‘‘Profile of Phillip Cohen,’’ by Carrie Arnold, Correction for ‘‘Origin, antiviral function and evidence for which appeared in issue 12, March 24, 2009, of Proc Natl Acad positive selection of the gammaretrovirus restriction gene Fv1 in Sci USA (106:4581–4583; first published March 17, 2009; the genus ,’’ by Yuhe Yan, Alicia Buckler-White, Kurt 10.1073/pnas.0902168106). Wollenberg, and Christine A. Kozak, which appeared in issue 9, The editors wish to note that because of several factual and March 3, 2009, of Proc Natl Acad Sci USA (106:3259–3263; first typographical errors we are retracting this article. A corrected published February 12, 2009; 10.1073͞pnas.0900181106). version will be published in a subsequent issue. The PNAS The authors note that due to a printer’s error, the database editors deeply regret the errors. accession numbers in the footnote on page 3259 appeared

͞ ͞ ͞ ͞ incorrectly. The footnote ‘‘Data deposition: The sequences www.pnas.org cgi doi 10.1073 pnas.0903490106 reported in this paper have been deposited in the GenBank database (accession nos. X97719, FJ603554, and X97720),’’ should instead have appeared as: ‘‘Data deposition: The se- quences reported in this paper have been deposited in the GenBank database (accession nos. FJ603554–FJ603574).’’ Ad- ditionally, on page 3259, left column, the first line of the third full paragraph, ‘‘Fv1 was cloned and identified as a coopted ERV sequence that is related to the gag gene of MuERV-L (3, 4), a Class III (spumavirus-related) ERV transposit family that is transpositionally active in mice but has no known infectious virus counterparts,’’ should instead have appeared as ‘‘Fv1 was cloned and identified as a coopted ERV sequence that is related to the gag gene of MuERV-L (3, 4), a Class III (spumavirus-related) ERV family that is transpositionally active in mice but has no known infectious virus counterparts.’’ These errors do not affect the conclusions of the article.

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MEDICAL SCIENCES Correction for ‘‘A genomic approach to colon cancer risk stratification yields biologic insights into therapeutic opportu- nities,’’ by Katherine S. Garman, Chaitanya R. Acharya, Elena Edelman, Marian Grade, Jochen Gaedcke, Shivani Sud, William Barry, Anna Mae Diehl, Dawn Provenzale, Geoffrey S. Gins- burg, B. Michael Ghadimi, Thomas Ried, Joseph R. Nevins, Sayan Mukherjee, David Hsu, and Anil Potti, which appeared in issue 49, December 9, 2008, of Proc Natl Acad Sci USA (105:19432–19437; first published December 2, 2008; 10.1073͞ pnas.0806674105). The authors note that the term ‘‘prognostic score’’ should be substituted for the term ‘‘Recurrence Score,’’ which is a regis- tered trademark of Genomic Health and is not associated in any way with the authors or the article. The online version of the article has been corrected accordingly as of April 7, 2009.

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6878 ͉ PNAS ͉ April 21, 2009 ͉ vol. 106 ͉ no. 16 www.pnas.org Downloaded by guest on September 24, 2021 Origin, antiviral function and evidence for positive selection of the gammaretrovirus restriction gene Fv1 in the genus Mus

Yuhe Yana, Alicia Buckler-Whitea, Kurt Wollenbergb, and Christine A. Kozaka,1 aLaboratory of Molecular Microbiology, bBioinformatics and Computational Biosciences Branch, Office of Cyber Infrastructure and Computational Biology, National Institute of Allergy and Infectious Diseases, Bethesda, MD 20892-0460

Communicated by Malcolm A. Martin, National Institutes of Health, Bethesda, MD, January 7, 2009 (received for review November 17, 2008) The Fv1 virus resistance gene is a coopted endogenous retrovirus reverse transcription and before integration. Fv1 is known to (ERV) sequence related to the gag gene of the MuERV-L ERV family. target the virus capsid gene; a single amino acid substitution at Three major Fv1 resistance alleles have been identified in labora- position 110 distinguishes N- and B-tropic viruses (7), and tory mice, and they target virus capsid genes to produce charac- substitutions at additional residues in the capsid N-terminal teristic patterns of resistance to mouse leukemia viruses (MLVs). domain are responsible for NR- and NB-tropism (5, 8). We identified Fv1 in 3 of the 4 Mus subgenera; its absence from Until recently, Fv1-type restriction had only been identified in and 1 of 3 species of indicate Fv1 was acquired laboratory mice and wild mouse species closely related to shortly after the origin of the Mus genus. We sequenced Fv1 genes laboratory mice (2). Our examination of an African pygmy from 21 mice representative of the major taxonomic groups of mouse, subgenus , identified an unusual postentry Mus. Two lines of evidence indicate that Fv1 has had antiviral resistance to ecotropic MuLVs (9). Although this resistance function for 7 million years of evolution. First, 2 species of African targets some of the same amino acid residues as the mouse Fv1 pygmy mice (subgenus Nannomys) show an Fv1-like MLV resis- gene, the pattern of virus resistance in the pygmy mouse cells tance, and transduced cells expressing the Nannomys Fv1 gene does not resemble that attributed to any of the reproduce this resistance pattern. Second, sequence comparisons EVOLUTION suggest that Fv1 has been involved in genetic conflicts throughout Fv1 alleles. Mus evolution. We found evidence for strong positive selection of We have now screened additional Mus species distantly related Fv1 and identified 6 codons that show evidence of positive selec- to laboratory strains for Fv1-like resistance phenotypes, and tion: 3 codons in the C-terminal region including 2 previously analyzed the Fv1 sequence in wild mouse species of 3 Mus shown to contribute to Fv1 restriction in laboratory mice, and 3 subgenera. We show here that the pygmy mouse Fv1 has antiviral codons in a 10-codon segment overlapping the major homology activity and demonstrate that Fv1 has been under strong positive region of Fv1; this segment is known to be involved in capsid selection throughout 7 MY of Mus evolution. We identified 6 multimerization. This analysis suggests that Fv1 has had an anti- codons under strong positive selection including 2 residues viral role throughout Mus evolution predating exposure of mice to implicated in Fv1-mediated virus restriction, and 3 codons in a the MLVs restricted by laboratory mouse Fv1, and suggests a segment overlapping the Fv1 major homology region (MHR) mechanism for Fv1 restriction. region, a region that in retroviruses produces the interface for capsid binding and dimerization. Fv1 gammaretrovirus restriction gene ͉ mouse endogenous retrovirus ͉ Mus evolution Results Analysis of the 4 Subgenera of Mus for Fv1. We examined repre- ild mouse species and inbred laboratory strains vary in sentative species of all 4 Mus subgenera (Mus, Pyromys, Nanno- Wtheir susceptibility to gammaretrovirus infection, and mys and Coelomys) for Fv1 sequences by Southern blot analysis such resistance can be due to constitutively expressed antiviral using a probe from the 5Ј end of Fv1 (Fig. 1A). One Fv1-reactive factors that target various stages of the retroviral life cycle. The band was detected in most species; Mus mus caroli has 2 prototype for such virus resistance factors is the Fv1 gene, BglI-generated Fv1-related copies (Fig. 1B). Fv1 was identified discovered 40 years ago in studies on resistance to Friend murine in3ofthe4Mus subgenera; it was missing in Mus coelomys pahari leukemia virus (MLV) (1). and in Mus pyromys shortridgei,1of3Pyromys species tested. There are 4 well characterized functional variants of Fv1 and PCR using primers designed to amplify the 3Ј end of Fv1 along additional Fv1-like restrictions found in inbred strains and wild with flanking sequences confirmed the absence of Fv1 from n b mouse species (2). Three of these alleles, termed Fv1 , Fv1 , and these 2 species (Fig. 1C). These primers also distinguish the 2 nr Fv1 , produce characteristic patterns of resistance to subgroups major Fv1 variants found in laboratory mouse strains; Fv1n has of mouse-tropic viruses that are designated N-, B-, or NR-tropic. a 1.3-kb deletion at its 3Ј end relative to Fv1b. Both Fv1 variants 0 Cells with the Fv1 (null) allele restrict none of these virus were identified in Mus species. Most mice carry the 1.3-kb subgroups, and NB-tropic viruses are not restricted by any of segment characteristic of Fv1b; the Fv1n deletion was found only Fv1 these alleles. in species, specifically all 4 Mus mus musculus Fv1 was cloned and identified as a coopted ERV sequence that is related to the gag gene of MuERV-L (3, 4), a Class III (spumavirus-related) ERV transposit family that is transposi- Author contributions: C.A.K. designed research; Y.Y. and A.B.-W. performed research; tionally active in mice but has no known infectious virus coun- A.B.-W. and K.W. analyzed data; and C.A.K. wrote the paper. terparts. The major resistance variants of Fv1 differ from one The authors declare no conflict of interest. b another at 3 amino acid sites in its C-terminal region, and Fv1 Data deposition: The sequences reported in this paper have been deposited in the GenBank n nr additionally differs from Fv1 and Fv1 at its C terminus due to database (accession nos. X97719, FJ603554, and X97720). a 1.3-kb indel (3). Substitutions at the 3 sites and variation at the 1To whom correspondence should be addressed. E-mail: [email protected]. C terminus all contribute to resistance (5, 6). The mechanism of This article contains supporting information online at www.pnas.org/cgi/content/full/ resistance is unknown, but Fv1 typically blocks replication after 0900181106/DCSupplemental. www.pnas.org͞cgi͞doi͞10.1073͞pnas.0900181106 PNAS Early Edition ͉ 1of5 Fig. 2. Comparison of predicted amino acid sequence of the M. n. minu- toides Fv1 gene and Fv1b. The position of MHR is indicated by a solid bar and shading indicates the 3 amino acids identified as critical for restriction based on analysis of laboratory mouse Fv1 alleles (3, 5, 6).

Fig. 1. Detection of Fv1 in DNAs of Mus species. (A) The structure of Fv1b is (Fv1n), 129/J (Fv1nr) and M. dunni (Fv10) (Table 1). M. pyromys shown with a gray box marking the MHR, open boxes representing B2 repeats platythrix was fully susceptible to all viruses tested indicating that and a dashed line representing the 1.3-kb segment deleted in Fv1n. The arrows represent the PCR primers Fb3003 and Rb4831, and the black box represents its Fv1 gene has no antiviral activity against this particular panel the segment used for blot hybridization. Fv1 is encoded by a single exon. (B) of MLVs. Cells of a second species, Mus nannomys minutoides, Southern blot analysis of BglI-digested Mus DNAs. All lanes taken from the were completely resistant to all AKV MLVs, but susceptible to same exposure of a single blot; deleted lanes are indicated by vertical lines. (C) MoMLV and Friend MLVs, as reported (9). In contrast, cells of PCR products of Mus DNAs. another Nannomys species, Mus nannomys setulosus, show low, but detectable susceptibility to AKV-B and AKV-NR, reduced susceptibility to MoMLV, and differential susceptibility to the 2 samples tested and some Mus mus domesticus and Mus mus Friend MLVs. M. n. setulosus cells, like M. n. minutoides, are spretus mice. completely resistant to infection with AKV-N and to an MoMLV Mus originated 7–8 MYA and quickly radiated into 4 subgen- chimera, Mo-CA3, in which part of the Fv1 target region (codon era. The radiations leading to these subgenera are difficult to positions 99–129) of the capsid gene was replaced with that of order, but Coelomys is generally regarded as the most basal AKV-N (9). Thus, these 2 pygmy mice have similar but group in Mus (10). Our results indicate that Fv1 is absent from distinctive patterns of susceptibility to MLVs, and their resis- species in 2 of the non-Mus subgenera, including Coelomys, and tance to AKV-N is mediated by the region of capsid that is that when Fv1 entered the Mus germ line it contained the 1.3-kb targeted by Fv1. segment found in the laboratory mouse Fv1b allele. Fv1 gene of M. n. minutoides and M. n. setulosis. The 5Ј and 3Ј ends Restriction of Ecotropic MLVs in Cells of 2 Species of Nannomys. To of the Fv1 gene of M. n. minutoides, Fv1m, were amplified from determine if Fv1 sequences serve an antiviral function in species genomic DNA and sequenced (Fig. 2); this sequence included a from non-Mus subgenera, we infected cells from these mice with complete 1.3-kb Fv1-related ORF and flanking sequences on various viruses known to be subject to restriction by laboratory both sides of the gene. The 24-bp segment flanking the 5Ј end mouse Fv1, and susceptibility was compared with NIH 3T3 is identical to that flanking Fv1b. The 803-bp flanking the 3Ј end of the ORF are homologous to the Fv1b flanking segment, but also contain a 170-bp B1 repeat inserted near the end of the Table 1. Virus titers of MLVs on cells of various Mus species and coding region. The 437-aa Fv1m ORF is highly homologous to the cells with known Fv1 restriction 440-aa Fv1 gene of the laboratory mouse. The Fv1m predicted Log10 Virus Titer*/Fv1 Tropism protein sequence is 87% identical to Fv1n. The differences AKV MLV MoMLV FBLV F-S MLV include a 9-base in-frame deletion near the N terminus, and 9-aa differences at the C terminus due to the B1 insertion. The rest Cells N B NR NB NB NR of the differences are scattered and notably include differences NIH 3T3 (Fv1n) 4.4 0.5 4.4 6.5 5.1 5.6 at the 3 amino acid positions (352, 358, and 399) that distinguish n b nr 129/J EF (Fv1nr) 1.8 0.9 4.2 5.8 ND 4.7 Fv1 , Fv1 , and Fv1 and have been shown to modulate resis- M. dunni (Fv10) 4.1 4.5 4.4 2.5† 4.1 4.7 tance (3, 5, 6). Residues at these positions for the known n b M. n. minutoides Ͻ0 Ͻ0 Ͻ0 4.5 4.3 4.0 restrictive Fv1 alleles are: SKV (Fv1 ), SER (Fv1 ), and FKV nr m M. n. setulosus Ͻ0 1.4 0.7 3.6 2.8 1.1 (Fv1 ). The Fv1 sequence has a different combination at these M. p. platythrix 3.7 4.4 4.4 5.8 4.1 4.4 sites, FKS. The predicted amino acid sequence of the Fv1 gene cloned *Virus titers were determined by the XC overlay test in which the indicated from M. n. setulosus cells, Fv1s, is 98% identical to Fv1m, and has cells were infected with virus dilutions, irradiated 4 days later and overlaid with XC cells to identify clusters of virus infected cells (9, 11). Titers represent a similar C terminus and the same 9-bp deletion in the N n s the number of XC PFU in 0.2 ml in representative experiments. ND, not done. terminus relative to Fv1 . For Fv1 , the amino acids at the 3 sites †Restriction is mediated by receptor polymorphism (12), and is unrelated to that distinguish the 3 known restrictive alleles are FKE, a unique Fv1. pattern.

2of5 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0900181106 Yan et al. Table 2. Virus titers of MLVs on MDTF cells transduced with Fv1n or Fv1m

Log10 Virus Titer*

Cells AKV-N AKV-B FBLV

MDTF 4.4 3.4 5.8 MDTF-Fv1n 4.7 0.8 5.4 MDTF-Fv1m 2.7 1.5 5.3

*Virus titers determined as in Table 1.

Functional Analysis of Fv1m. Full length Fv1 ORFs from M. n. minutoides cells and from NIH 3T3 were expressed in MDTF cells (Fv10) (13). Cells were tested for susceptibility to viruses restricted by NIH 3T3 and by M. n. minutoides cells (Table 2). The transduced cells were fully susceptible to NB-tropic Friend MLV (FBLV), but MDTF cells expressing Fv1n showed reduced susceptibility to AKV-B, and cells expressing Fv1m showed reduced susceptibility to both AKV-B and AKV-N. The fact that the transduced cells did not reproduce the level of restriction seen in M. n. minutoides or NIH 3T3 cells is consistent with previous observations that Fv1 is highly sensitive to concentra- tion and that not all transfected cells show restriction (3, 14). In 3 independent experiments, however, the transduced MDTF cells reproduced the distinctive restriction patterns of M. n. minutoides and NIH 3T3. EVOLUTION

Fv1 Evolution. Host genes involved in antagonistic interactions with pathogens can be identified by sequence comparisons that reveal evidence of positive selection. To determine if Fv1 has had an antiviral role throughout Mus evolution, and to identify Fig. 3. Positive selection of Fv1 in the genus Mus.(A) Cladogram showing possible sites of virus interaction, we analyzed 24 sequenced Fv1 branch values of dN/dS calculated using the free-ratio model of PAML, with the number of replacement and synonymous changes in parentheses. When genes from various Mus species. dS ϭ 0, dN/dS is infinite (Inf). dN/dS Ͼ 1 suggests positive selection along that Two of the Fv1 genes, in M. mus dunni and M. mus cookii, had lineage. Bootstrap support for this tree topology was generally good, with stop codons in the 5Ј half of the gene as found in a previous most bootstrap percentages Ͼ90%. The thin lines represent branches with analysis of this segment of the Fv1 sequence (15); these were bootstrap values Ͻ70%. (B) Likelihood ratio tests were used to test for positive excluded from further analysis. The near full-length Fv1 se- selection. Neutral models (M0, M1, M7) were compared with selection models quences used for selection analysis did not include gaps in the (M2, M8) using 2 different models of codon frequency (F3 ϫ 4 or F61). P values sequence, and codons after the stop codon in M. nannomys triton Ͻ0.0001 provide strong evidence of selection. Tree length is the average (Dataset S1). All of the sequenced Fv1 genes were amplified number of substitutions per codon along all branches. For codons under using one or more primers designed from the sequences flanking positive selection, the dN/dS ratio is given along with the % of codons with this ratio. the Fv1b ORF, indicating that these genes represent the same integration site. Twenty-two Fv1 genes were used to construct a maximum parsimony tree (Fig. 3A). Values of dN/dS along each contribute to virus restriction (5, 6). A third codon, 358, previ- branch were calculated using the free-ratio model of PAML (16). ously implicated in Fv1 restriction in laboratory mice was not Ͼ A dN/dS value 1 suggests that positive selection has acted along identified as being under positive selection; all Fv1 genes except that lineage. Many branches of the tree show evidence of positive b Ͼ ϭ for Fv1 have K at this site suggesting that the laboratory mouse selection with dN/dS 1, or, when dS 0, show 4 or more Fv1b variant K358E arose fairly recently in Mus. The demon- replacement substitutions, which is also suggestive of selection. stration of positive selection at these 6 codons suggests that the Likelihood ratio tests indicate that Fv1 has a significant Fv1 gene has been subjected to positive selection over nearly 7 probability of having experienced positive selection (Fig. 3B and MY of Mus evolution, and indicates that residues known to be SI Appendix); this is consistent with a previous analysis of a responsible for restriction of MLVs in laboratory mice also have smaller segment of Fv1 (15). The Bayes empirical Bayes calcu- lation of posterior probabilities in PAML (17) identified specific antiviral activity in more evolutionarily divergent species and Fv1 codon positions as having significant probability of positive subgenera. selection. Results identified 6 codons as being under positive Discussion selection with posterior probability Ͼ0.95 (Fig. 4), and for all 6 codons P Ն 0.99. A second analysis done with an equally These results indicate that the mouse Fv1 gene has had antiviral parsimonious PAUP-generated tree identified the same 6 activity since shortly after its introduction into the Mus genome, codons with nearly identical statistics. The 6 codons identified as an event that occurred during a period of rapid diversification being under positive selection included 3 at positions 261, 265, that gave rise to the 4 Mus subgenera Ϸ7 MY ago (10, 23). That and 270, clustered at the 5Ј end of the MHR, a highly conserved this sequence has long had antiviral function is supported by the region known to be involved in capsid interactions (18–22). The observation that the Fv1 genes of 2 species of Nannomys have other 3 codons identified as being under positive selection were novel Fv1-mediated antiviral phenotypes, and by the demon- at positions 352, 399, and 401; 2 of these codons, 352 and 399, stration that Fv1 has been under positive Darwinian selection. distinguish the laboratory mouse Fv1 restriction genes (3, 5), and We identified 2 segments of Fv1 potentially involved in restric- substitutions at both of these codons have been shown to tion: an MHR segment known to function in capsid binding, and

Yan et al. PNAS Early Edition ͉ 3of5 diversification Ϸ7 MYA; this is supported by the observation that 2 species from 2 of the 4 subgenera that originated at that time lack Fv1. Evidence of positive selection of the Fv1 gene, the identification of 2 different but related Fv1-like restriction genes in Nannomys and the demonstration that one of these cloned Fv1 genes, Fv1m, reproduces this resistance phenotype in transduced cells suggests Fv1 has had an antiviral role throughout Mus evolution. The exposure of mice to MLVs is marked by the appearance of related ERVs, but the appearance of Fv1 in Mus significantly predates the acquisition of ERVs of MLVs, the viruses restricted by Fv1. We demonstrated that MLV ERVs are largely found only in house mouse species that appeared only Ϸ1 MYA, that is, M. m. domesticus, castaneus and musculus (24). Our evolutionary and functional analysis of Fv1 in other species and subgenera uncovered evidence of antiviral activity in MLV-free species, which suggest that Fv1 may have broader antiretroviral activity than previously appreciated. Materials and Methods Cells and Viruses. Ecotropic (mouse-tropic) MLV isolates were obtained from Fig. 4. Fv1 sites that have been subject to positive selection. At the top are J. W. Hartley (National Institute of Allergy and Infectious Diseases, Bethesda, sequence alignments for 22 Mus genes in the regions containing the 6 posi- MD) and included AKV MLV (AKV-N), WN1802B (AKV-B), AKR-L1 (AKV-NR), tively selected codons; all changes relative to Fv1b are shaded. MHR is boxed. Moloney MLV (MoMLV), and 2 Friend MLV (FrMLV) isolates, NR-tropic F-S MLV At the bottom is a diagram of the Fv1 coding region showing the locations of and NB-tropic FBLV (9). Mo-CA3 is a chimera of MoMLV with a segment of AKV the MHR (black box), the 6 positively selected codons and the 1 additional MLV capsid containing the Fv1 target region between residues 99 and 129 (9). codon (358) implicated in restriction (3, 6). Virus stocks were made by collecting culture fluids from virus infected cells. The XC overlay test (9, 11) was used to test for susceptibility to virus infection in various cell lines including 2 lines derived from the Asian species 3 residues in the C-terminal domain (CTD) of which 2 are known Mus m. dunni, MDTF and M. dunni (13), NIH 3T3 and cell lines derived from tail to contribute to the specificity of Fv1 restriction in laboratory mice. fibroblasts of the wild mouse species M. n. minutoides, M. n. setulosus, and M. The mechanism of Fv1 restriction has not been elucidated p. platythrix obtained from J. Rodgers (Baylor College of Medicine, Houston, after 40 years of investigation, but the determination that Fv1 TX) (25). Embryo fibroblasts were prepared from strain 129/J from The Jackson Laboratory. encodes a retroviral capsid-like protein suggests that Fv1 may bind capsids of exogenous virus and interfere with capsid Wild Mouse Genomic DNA. DNA was isolated from the wild mouse-derived cell disassembly and reverse transcription. Three of the positively lines listed above, from individual NFS/N mice from our colony and from wild selected codons identified in this study are in a 10-codon mice from various sources (Table S1). Most mice were originally obtained from segment associated with the Fv1 capsid-like MHR region. Mu- M. Potter (National Cancer Institute, Bethesda, MD). A set of African pygmy tations of codons in and around the MHR regions of various mouse DNA samples was obtained from Y. Cole and P. D’Eustachio (New York retroviruses disrupt virus assembly, maturation and infectivity University, New York); these mice had been classed into 4 species of Nannomys (18–20). Mutational analysis in and around the MHR of mice on the basis of skeletal features by J. T. Marshall (Smithsonian Natural MoMLV and HIV-1 has determined that the segment described History Museum, Washington, DC). A sample of M. m. macedonicus DNA was here is critical for formation of the high-affinity capsid interface, provided by R. Elliott (Roswell Park, Buffalo, NY), and M. m. domesticus mice trapped in California were provided by S. Rasheed (University of Southern and that this segment determines the specificity of heterodimeric California, Los Angeles). M. m. spretus (SPRET/EiJ), M. m. castaneus (CAST/EiJ), interactions (20, 21). These interactions are important in the M. m. molossinus, BALB/cJ, and C57BL/6J were obtained from The Jackson early stages of replication as the majority of replication defective Laboratory. All protocols were reviewed and approved (Proposal no. MoMLV CA mutants are blocked before reverse transcription LMM1) by the National Institute of Allergy and Infectious Diseases Animal (22). Mutations within this segment of the Fv1 MHR demon- Care and Use Committee. strate this region is critical for Fv1 restriction (6), but while All wild-derived mice are named using genus, subgenus, and species. studies on Fv1 have not produced direct evidence for Fv1-capsid binding, our results support an antiviral model based on Fv1- Cloning and Functional Analysis of M. n. minutoides Fv1. The 5Ј end of the Fv1 capsid interactions. gene of M. n. minutoides (Fv1m) was PCR amplified and sequenced using b Three of the 6 positively selected residues include 2 in the CTD primers based on Fv1 and numbered according to GenBank accession no. X97719: Fb2136-AGCCGAGTTCTAGGGAAACAA and Rb3133-TCAGCCAAC- that have already been identified as critical for the specificity of Ј m n nr CAATCAAACAAACT. The 3 end of Fv1 was then amplified from NcoI di- Fv1 restriction. F/S352 distinguishes Fv1 and Fv1 restriction gested DNA using inverse PCR (26) with Fv1m-based primers from GenBank types, and substitutions at codon 399 alter restriction specificity accession no. FJ603554: Fm568-ATAGATTCTGATGGGACTGAG and Rm184- (5, 6). Our analysis also identified positive selection at a position TACACAGAGTCATTAAGTTCCTTACC. The PCR product was sequenced. in this region not previously known to affect restriction, 401; its The Fv1m coding region was then amplified from total RNA of M. n. role in restriction may be defined by further mutational analysis. minutoides cells using primers with introduced HindIII and ClaI sites (Fm22- A 4th codon in this region of the CTD, 358, is known to affect TCAAGCTTAGGATGAATTTCCTACGTGCGCTTGCTG and Rm1313-TTATCGATT- MLV-N/B/NB restriction specificity (6), but we found this site to TAAAGGCCACCACGCCCGGCTTTTG) and cloned into the retroviral vector n be highly invariant in Mus species. Lack of sequence variation at pLNCX2 (Clontech). The full length Fv1 gene was similarly amplified from this site suggests that the only known substitution at the 358 total RNA extracted from NIH 3T3 cells with primers based on GenBank b accession no. X97720: Fn35-TCAAGCTTAGGATGAATTTCCCACGTGCGC- codon identified to date, in the Fv1 laboratory mouse strains, TTGCTG and Rn1332-TTATCGATTCAGAGTTTTGTAGCTGCTGTTGGCT and likely emerged very recently in Mus evolution, perhaps in cloned into pLNCX2. The Fv1 genes were packaged by cotransfecting GP2–293 response to the emergence of the N-tropic MLV it restricts. cells (Clontech) with the Fv1 clones or empty vector and pVSV-G (Clontech). Fv1 is not found in all mice. Fv1 entered the mouse germ line Culture fluids were collected and used to infect MDTF cells, and stably trans- shortly after the origination of Mus duringa1MYperiod of duced cells were selected for virus infection.

4of5 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0900181106 Yan et al. Fv1 Sequences of Other Wild Mouse Species. BglI digested DNAs were screened branch-specific rates of dN/dS. In this model each branch is assumed to have a for the presence of Fv1 by Southern blot analysis using as probe a 358-bp specific dN/dS ratio. The likelihood of the phylogeny under this model was segment of the 5Ј end of Fv1b amplified from C57BL/6 DNA using primers tested against the likelihood of the phylogeny under the model of one Fb2165-AGATGAATTTCCCACGTGC and Rb2497-AGGACACACTTAGAAGC- uniform dN/dS ratio across all branches (codon model 0) using a likelihood CTTTAGATC. This segment shows 27% identity to the MuERV-L family of ratio test (LRT). The significance of the LRT value was assessed using a ␹2 which Fv1 is a member (4). distribution with 36° of freedom. Fv1 was amplified from various mouse genomic DNAs using primers de- Selection acting on Fv1 codons was analyzed using 2 models of equilibrium signed from coding and flanking sequences of Fv1m and Fv1b. Flanking se- codon frequencies and 4 models of codon selection. The 2 codon frequency quence primers included Fb2136-AGCCGAGTTCTAGGGAAACAA, Rb3703- models used were the F3 ϫ 4 model (codon frequencies estimated from the TTTGCAACCAACCAGTGGCA, Rb4179-TCATAGCATATGTGAACAATCA, and nucleotide frequencies in the data at each codon site) and the F61 codon table Rb4831-CATCTATACTATCTTGGTGAG. From Fv1 coding sequences, we used model (frequencies of each of the 61 non-stop codons estimated from the primers Fb2165, F/Rb3003-TTTAAGGGTGTGGGATAATGGT and F/Rb3133- data). The codon selection models were 2 neutral/negative selection models AGTTTGTTTGATTGGTTGGCTGA. Most Fv1 genes were sequenced as 2 over- (M1 and M7), which were compared against corresponding models including lapping PCR products, some were first cloned into pCR2.1-TOPO (Invitrogen, a category for dN/dS Ͼ1 (M2 and M8, respectively). The significance of this Carlsbad, CA) before sequencing (Dataset S1). additional codon selection category was assessed using LRTs of the phylogeny likelihoods under the neutral and positive selection models. Significance of Selection Analysis of Lineages and Codons. DNA sequences were aligned using the test statistics was calculated using a ␹2 distribution with 2 degrees of MUSCLE (27) and improved manually. The phylogeny used was 1 of 4 equally freedom. The Bayes empirical Bayes algorithm (17) was used to calculate the parsimonious phylogenies returned by PAUP* (version 4.0b10) (28), and was posterior probability of individual codons experiencing dN/dS Ͼ1. chosen by correspondence to consensus Mus phylogeny (10, 23). Two of the 4 equally parsimonious trees differed only in the arrangement of zero-length ACKNOWLEDGMENTS. We thank Esther Shaffer and Qingping Liu for expert branches, leaving only 2 different tree topologies. The codeml program of the technical assistance. This work was supported by the Intramural Research PAML4 package (16) was used for maximum likelihood analysis of codon Program of the National Institutes of Health, National Institute of Allergy and evolution (29). The free ratio model (codon model ϭ 1) was used to calculate Infectious Diseases.

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