Nova Southeastern University NSUWorks Biology Faculty Articles Department of Biological Sciences 4-2005 Insertional Polymorphisms of Endogenous Feline Leukemia Viruses Alfred L. Roca National Cancer Institute at Frederick William G. Nash National Cancer Institute at Frederick Joan C. Menninger National Cancer Institute at Frederick William J. Murphy National Cancer Institute at Frederick; Texas A&M University - College Station Stephen J. O'Brien National Cancer Institute at Frederick, [email protected] Follow this and additional works at: https://nsuworks.nova.edu/cnso_bio_facarticles Part of the Animal Sciences Commons, Genetics and Genomics Commons, Veterinary Medicine Commons, and the Virology Commons NSUWorks Citation Roca, Alfred L.; William G. Nash; Joan C. Menninger; William J. Murphy; and Stephen J. O'Brien. 2005. "Insertional Polymorphisms of Endogenous Feline Leukemia Viruses." Journal of Virology 79, (7): 3979-3986. https://nsuworks.nova.edu/cnso_bio_facarticles/ 206 This Article is brought to you for free and open access by the Department of Biological Sciences at NSUWorks. It has been accepted for inclusion in Biology Faculty Articles by an authorized administrator of NSUWorks. For more information, please contact [email protected]. JOURNAL OF VIROLOGY, Apr. 2005, p. 3979–3986 Vol. 79, No. 7 0022-538X/05/$08.00ϩ0 doi:10.1128/JVI.79.7.3979–3986.2005 Copyright © 2005, American Society for Microbiology. All Rights Reserved. Insertional Polymorphisms of Endogenous Feline Leukemia Viruses Alfred L. Roca,1* William G. Nash,2 Joan C. Menninger,1 William J. Murphy,1,3 and Stephen J. O’Brien4* Laboratory of Genomic Diversity, Basic Research Program, SAIC-Frederick,1 and Laboratory of Genomic Diversity, National Cancer Institute,4 Frederick, Maryland; H&W Cytogenetic Services, Inc., Lovettsville, Virginia2; and Department of Veterinary Integrative Biosciences, Texas A&M University, College Station, Texas3 Downloaded from Received 1 September 2004/Accepted 10 December 2004 The number, chromosomal distribution, and insertional polymorphisms of endogenous feline leukemia vi- ruses (enFeLVs) were determined in four domestic cats (Burmese, Egyptian Mau, Persian, and nonbreed) using fluorescent in situ hybridization and radiation hybrid mapping. Twenty-nine distinct enFeLV loci were detected across 12 of the 18 autosomes. Each cat carried enFeLV at only 9 to 16 of the loci, and many loci were heterozygous for presence of the provirus. Thus, an average of 19 autosomal copies of enFeLV were present per cat diploid genome. Only five of the autosomal enFeLV sites were present in all four cats, and at only one http://jvi.asm.org/ autosomal locus, B4q15, was enFeLV present in both homologues of all four cats. A single enFeLV occurred in the X chromosome of the Burmese cat, while three to five enFeLV proviruses occurred in each Y chromosome. The X chromosome and nine autosomal enFeLV loci were telomeric, suggesting that ectopic recombination between nonhomologous subtelomeres may contribute to enFeLV distribution. Since endogenous FeLVs may affect the infectiousness or pathogenicity of exogenous FeLVs, genomic variation in enFeLVs represents a candidate for genetic influences on FeLV leukemogenesis in cats. on January 14, 2016 by NOVA SOUTHEASTERN UNIV Endogenous feline leukemia virus (enFeLV) sequences are enFeLVs in vivo (7, 26). Likewise, inoculation with recombi- present in the genome of the domestic cat, Felis catus. They are nant subgroup B exogenous FeLV attenuates infection by sub- homologous to exogenous feline leukemia viruses, which are group A exogenous FeLV (45). By contrast, a protein derived oncogenic retroviruses capable of inducing both proliferative from an enFeLV env region was found to facilitate infection by and degenerative diseases (15, 34). Whereas exogenous FeLVs a T-cell-tropic exogenous FeLV (2). are transmitted horizontally, endogenous feline leukemia pro- The number of copies of enFeLV per haploid genome has viruses are part of the germ line and are transmitted from been estimated as 6 to 12 (5, 20, 39, 41, 42), arranged in a parent to offspring as integral components of chromosomes (5, nontandem manner (20), while the number of freestanding 20). Endogenous FeLVs are found in wild species of the genus long terminal repeats (LTRs), believed to have lost their as- Felis closely related to the domestic cat, although they are not sociated coding regions through unequal crossing over during present in species from other lineages within the Felidae (4, recombination, is ϳ150 (8). Despite the biomedical impact of 56–58). Thus, enFeLVs are believed to have entered the germ feline leukemia viruses and the established capacity of endog- line after the initial radiation of lineages in the cat family but enous feline leukemia viruses to recombine with exogenous before the radiation of domestic cat lineage species (3, 23, 25), virus to influence infection and disease progression, the geno- although subsequent additional integrations into the germ line mic distribution and variation of enFeLVs among domestic may also have occurred (50). cats have not been well characterized. Here, we used fluores- Endogenous FeLV sequences do not by themselves produce cent in situ hybridization (FISH) and radiation hybrid (RH) infectious virus but readily recombine with exogenous feline mapping to determine the chromosomal locations and in- leukemia viruses, notably in the env region that codes for the traspecies variation of enFeLVs in four domestic cats. viral coat, producing recombinant viruses with altered biolog- ical activity and pathogenicity (4, 17, 21, 43, 44, 51, 53, 54, 60). MATERIALS AND METHODS For example, the recombinant subgroup C viruses have been Probe generation and library screening. To generate probe for screening, long found to induce aplastic anemia (18). Furthermore, portions of PCR (see below) was performed using the DNA template pKHR-2/␭HF60 (13, the enFeLV env region are transcribed and translated in lym- 33), containing a full-length subgroup B recombinant feline leukemia provirus, Ј Ј phoma and other cell lines (26), producing a truncated enve- using primers GA-GAG-F1 (5 -ATGGGCCAAACTATAACTACCC-3 ) and GA-ENV-R1 (5Ј-TGGTCGGTCCGGATCGTATTGC-3Ј). Thermal cycling lope protein that inhibits infection by exogenous subgroup B consisted of an initial 94°C for 4.5 min; 3 cycles of 94°C for 30 s, 66°C for 25 s, feline leukemia viruses (26). Transcription and translation of and 68°C for 9 min; 5 cycles of 94°C for 30 s, 63°C for 25 s, and 68°C for 9.5 min; enFeLVs have also been demonstrated in tissues from healthy 30 cycles of 94°C for 30 s, 60°C for 25 s, and 68°C for 10 min; and a final extension cats, including lymphoid tissue, suggesting a protective role for of 68°C for 20 min. This yielded a 7-kb fragment, designated pKHR2-gpe, containing the gag, pol, and env genes, but not the LTRs, of the provirus. The pKHR2-gpe DNA (50 ng) was used to generate probe labeled with [␣-32P]dCTP (New England Nuclear) by random priming (Pharmacia). The probe was eluted * Corresponding author. Mailing address: Laboratory of Genomic through a Nick G-50 Sephadex column (Pharmacia). Diversity, National Cancer Institute-Frederick, Building 560, Freder- A female cat lambda FIX II genomic library (9- to 23-kb insert size; Strat- ick, MD 21702-1201. Phone: (301) 846-1296. Fax: (301) 846-1686. agene) was serially diluted with salt-magnesium buffer for appropriate plating E-mail for A. L. Roca: [email protected]. E-mail for S. J. O’Brien: (12 plates; 100,000 PFU/plate) and screened with labeled pKHR2-gpe at low [email protected]. stringency, as described previously (47–49). Positive plaques were rescreened 3979 3980 ROCA ET AL. J. VIROL. until isolated and then plated for extraction of phage DNA (52). Two enFeLV the presence of enFeLV when signal was detected at a chromosomal site in integrations, enFeLV-AGTT (phage 6) and enFeLV-GGAG (phage 16 and 80), Ն10% of the metaphase spreads. have been described (50). A novel enFeLV sequence in phage isolate number 3 Statistical analysis. The total number of enFeLV integrations among the eight was designated enFeLV-ATGC (ATGC for a 4-bp cat genomic sequence at the copies of each autosome (two homologues for each autosome in each of four integration site that is duplicated on either side of the provirus; see below). The cats) was divided by the relative physical length of the autosome (30), and a enFeLV in phage 18 was designated enFeLV-CTCT, and the enFeLV in phage two-tailed t test was implemented in Microsoft Excel comparing the number of 94 was designated enFeLV-AGAG. enFeLVs per unit length among cat autosomes. Distinguishing enFeLV loci. Like other retroviruses (59), FeLV duplicates a RH mapping. RH mapping was performed in a 93-clone domestic cat 5,000- small section of the host genome at the integration site. Thus, the 4 bp of feline rad RH panel as previously described (35, 36). The primers used for radiation genomic DNA found immediately upstream of the enFeLV 5Ј LTR has the same hybrid mapping of the cat genomic regions flanking enFeLV integration sites were as follows: for enFeLV-AGTT, ENFELV6-F (5Ј-TGGGGGAAAACCTT sequence and orientation as the 4-bp feline genomic DNA sequence immediately Downloaded from downstream of the 3Ј LTR. This duplicated 4-bp feline genomic sequence varies ACCTTC-3Ј) and ENFELV6-R (5Ј-CCCCTTCAGTGCATACAACA-3Ј); for Ј Ј depending on the genomic site at which the enFeLV provirus integrated; thus, we enFeLV-GGAG, ENFELV16-F (5 -GGGCAATTTACCCACAGAGA-3 ) with Ј Ј use it to distinguish different enFeLV loci. For example, when the duplicated ENFELV16-R (5 -CCTCAGCTTTGTTCTACGGG-3 ); for enFeLV-CTCT, Ј Ј Ј sequence was AGTT, we referred to the corresponding provirus as enFeLV- PH18-F1 (5 -TGCATTTAATGGGGTCTTGG-3 ) with PH18-R1 (5 -TCTGTG Ј Ј AGTT (AGTT for the cat genomic DNA tetramer on either side of the inte- GGGGTGAATTTTTC-3 ); for enFeLV-ATGC, ENFELV3-F (5 -TTCCAGG Ј Ј grated provirus).