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Genetic Variation Among Lentiviruses: Homology JOURNAL OF VIROLOGY. Sept. 1984. p. 713-721 Vol. 51. No. 3 0022-538X/84/090713-09$02 .00/0 Copyright © 1984, American Society for Microbiology Genetic Variation Among Lentiviruses: Homology Between Visna Virus and Caprine Arthritis-Encephalitis Virus Is Confined to the 5' gag-pol Region and a Small Portion of the env Gene JOANNA M. PYPER, JANICE E. CLEMENTS,* SUSAN M. MOLINEAUX, AND OPENDRA NARAYAN Department of Nelorology, The Johns Hopkins University S( hool oJ Medicine, Btiltitnor-e, Mal-viltnd 21205 Received 27 February 1984/Accepted 4 June 1984 Visna virus of sheep and arthritis-encephalitis virus of goats are serologically related but genetically distinct retroviruses which cause slowly progressive diseases in their natural hosts. To localize homologous regions of the DNAs of these two viruses, we constructed a physical map of caprine arthritis-encephalitis virus DNA and aligned it with the viral RNA. Cloned probes of visna virus DNA were then used to localize regions of homology with the caprine arthritis-encephalitis virus DNA. These studies showed homology in the 5' region of the genome encompassing U5 and the gag and pol genes and also in a small region in the env gene. These findings correlate with biological data suggesting that the regions of the DNA which are homologous may be responsible for virus group characteristics such as the closely related virus core antigens. Regions which did not show homology such as large sections in the env gene may represent unique sequences which control highly strain- specific characteristics such as the neutralization antigen and specific cell tropisms. Lentiviruses of sheep and goats are a distinct group of noncytopathic persistent infection in which virus replication nononcogenic retroviruses which cause slowly progressive is restricted more than 1,000-fold (34). diseases involving the central nervous system, lungs, and To determine the genetic basis for the differences in host joints (8, 9, 12, 13, 17, 35, 43). The lesions are characterized responses to these two viruses, we have begun studies on the by persistent inflammation (active-chronic type) which even- nucleic acids of prototypes of these two virus groups, tually leads to paralysis, respiratory failure, and crippling focusing initially on regions of homology between their arthritis (8, 9, 10, 11). Sheep develop mainly the pneumonic DNAs. Previous hybridization studies have shown that there form of the disease complex, and this leads to neurological is only minor homology between the nucleic acids of visna complications in some animals (17, 35). In goats, the disease virus and CAEV (16, 38). However, the physical location of expression is age dependent with neurological disease occur- homologous and nonhomologous regions between the two ring mainly in young animals and arthritis occurring mainly viral genomes has not been determined. In this study, we in adults (8, 9, 10, 11). The sheep viruses, of which visna is constructed a restriction endonuclease map of the DNA of the prototype, were discovered during epizootics of pneu- CAEV and aligned it with the viral RNA. With cloned moencephalitis (maedi-visna) in Iceland in the 1940s (40. 41), fragments of visna virus DNA (28) and different degrees of and the caprine viruses were discovered during an outbreak stringency for hybridization, we localized regions of nucleic of arthritis and encephalitis among goats in Washington state acid homology and identified regions of the genomes of the in the 1970s (8, 10). These viruses are widespread in sheep two viruses which were distinct. These genetic differences and goat populations in many parts of the world. In experi- and similarities were then compared with the immunological ments, both viruses cause persistent infections and replicate specificities of viral polypeptides determined in immunopre- at a slow but continuous rate for an indefinite period. The cipitation tests and virus neutralization experiments. These continuous release of viral antigens in specific tissues stimu- studies provide new insights into the relationship between lates the inflammatory response which leads to disease. viral genes, gene products, and antiviral host responses and Visna virus and caprine arthritis-encephalitis virus suggest an experimental basis for evaluating the role of viral (CAEV) have different mechanisms for maintaining persis- genes in the outcome of these slow infections. tent replication in their immunocompetent hosts. Both virus- es induce binding antibodies to all their respective polypep- MATERIALS AND METHODS tides during infection. However, they differ greatly with respect to induction of neutralizing antibodies. CAEVs do Virus and cells. CAEV strain CO was used in this study. not induce neutralizing antibodies during natural or experi- The history and characterization of the agent have been mental infections, in contrast to visna viruses, which do so reported previously (8, 29, 33). The virus was plaque purified readily (24, 33). Neutralizing antibodies to visna viruses are and propagated in cultures of goat synovial membrane highly specific. However, the range of neutralization is too (GSM) cells as described earlier (29). Visna virus strain 1514 narrow to inhibit replication of mutant viruses which arise in was used in this study; the plaque-purified virus stock was the immune host. The result of this narrow neutralization grown in sheep choroid plexus cells (SCP) as previously range is selective replication of virus mutants in the persis- described (30, 32). tently infected animal (antigenic drift) (4, 30. 31). These two DNA. GSM or SCP cells were infected at a multiplicity of viruses also vary in their efficiency of replication in sheep infection of 0.5 to 1 and harvested at peak cytopathic effect fibroblasts. Visna viruses cause a highly productive lytic (5 to 7 days postinfection). Low-molecular-weight DNA was infection in these cultures. In contrast, CAEVs cause a isolated as described by Hirt (19), and the supernatant DNA was processed as described by Clements et al. (7) except that * Corresponding author. the RNase A digestion was for 30 min. and digestion with 50 713 714 PYPER ET AL. J. VIROL. pLg of proteinase K per ml-0.5% sodium dodecyl sulfate was Two probes for the U3 region were subcloned into M13mplO substituted for the pronase digestion. from the SstI clone 8.5 and included 8.9 to 9.7 kb (F) and 9.7 Enzymes. Restriction enzymes were purchased from Be- to 10.15 kb (G) from the 5' end of the visna virus DNA. A 5' thesda Research Laboratories, Rockville, Md., or New fragment (0.9 to 1.95 kb from the 5' end of the visna virus England BioLabs, Beverly, Mass., and were used under DNA) from the Sstl 8.5 clone of strain 1514 was also conditions suggested by Maniatis et al. (27). Other pur- subcloned into M13mplO (A). These subclones were labeled chased enzymes were reverse transcriptase (Life Sciences, by using the probe primer (P-L Biochemicals, Inc., Milwau- St. Petersburg, Fla.), RNase-A (Calbiochem-Behring, La kee, Wisc.) and the conditions described by Ricca et al. (36). Jolla, Calif.), and proteinase K (Boehringer-Mannheim Bio- The specific activities of these probes ranged from 7.4 x 106 chemicals, Indianapolis, Ind.). to 1.6 x 107 cpm/[lg. Virus purification. Cells (either GSM cells for CAEV or Agarose gel electrophoresis. DNA was analyzed by electro- SCP cells for visna virus) were infected at a multiplicity of phoresis in 1.2% 3-mm agarose gels, transferred to nitrocel- infection of 0.5 to 1 in 850-cm2 roller bottles (Corning Glass lulose by the method of Southern (42), and hybridized to Works, Corning, N.Y.), and supernatants from the infected different probes under conditions described below. cultures were collected 1 to 2 times per day for 5 to 6 days. Hybridization of probes to DNA transfers. For mapping After clarification of the supernatant fluid at 8,000 rpm in a studies, DNA transfers were hybridized in 50% formamide GSA rotor (Ivan Sorvall, Inc., Norwalk, Conn.) for 30 min, (FLUKA AG) containing 6x SSC, 20 jig of single-stranded the virus was concentrated (100-fold) with a pellicon mem- DNA per ml, 20 jig of tRNA per ml, 50 mM N-2-hydroxyeth- brane concentrator (Millipore Corp., Bedford, Mass.) and ylpiperazine-N'-2-ethanesulfonic acid (pH 7), 0.02% poly- then pelleted at 27,000 rpm in an AH627 rotor (Sorvall) at vinylpyrollidine, and 0.02% bovine serum albumin at 42°C 4°C. The virus was suspended in 0.1 M NaCl-1 mM EDTA- for 36 to 48 h. DNA transfers were washed for 15 min in 2x 20 mM Tris (pH 7.4), diluted 1:1 with 5.3 M NaCl-1 mM SSC at 23°C, 1 h in 1x SSC-0.1% SDS at 50°C, and 2 min in EDTA-20 mM Tris (pH 7.4) (45), incubated at 37°C for 30 1 x SSC at 230C. DNA transfers were air dried and exposed min, and diluted 1:1 with 20 mM Tris (pH 7.4)-i mM EDTA. to Kodak XAR-5 film at -70°C with intensifying screens. Virus was banded by centrifugation for 2 h at 20,000 rpm in a Hybridization conditions used in homology studies were Sorvall SS-90 at 4°C on a step gradient of 55 to 20% sucrose similar to those described by Howley et al. (20). DNA in 1 M NaCI-1 mM EDTA-20 mM Tris (pH 7.4). Virus was transfers were hybridized as for mapping except that the pelleted at 27,000 rpm in an AH627 rotor (Sorvall) and formamide concentration was varied, and hybridization was suspended in 0.1 M NaCl-1 mM EDTA-20 mM Tris (pH 7.4) performed at 350C. Preliminary washes were in 2x SSC for (2,000-fold concentration of original supernatant).
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