Reactivation of Feline Foamy Virus from a Chronically Infected Feline Renal Cell Line by Trichostatin A

Virology 283, 315–323 (2001) doi:10.1006/viro.2000.0861, available online at http://www.idealibrary.com on View metadata, citation and similar papers at core.ac.uk brought to you by CORE

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Shinichi Hatama,* Kaori Otake,† Michio Ohta,‡ Miya Kobayashi,§ Kazuhiko Imakawa,¶ Atsushi Ikemoto,࿣ Harumi Okuyama,࿣ Masami Mochizuki,** Takayuki Miyazawa,* Yukinobu Tohya,* Yoichi Fujii,࿣ and Eiji Takahashi*,1

*Department of Veterinary Microbiology, ¶Laboratory of Animal Breeding, Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo; †Department of Immunology, National Institute of Infectious Disease, Tokyo; ‡Department of Bacteriology and §Department of Anatomy, Nagoya University School of Medicine, Nagoya; ࿣Department of Biological Chemistry, Faculty of Pharmaceutical Sciences, Nagoya City University, Nagoya; and **Central Laboratory, Kyoritsu Shouji Co., Ltd., Ibaraki, Japan Received October 31, 2000; returned to author for revision January 17, 2001; accepted February 7, 2001

Although acute infection of feline foamy virus (FeFV) is normally highly cytopathogenic in Crandell feline kidney (CRFK) cells, a noncytopathic persistent infection was established in the cells after cocultivation of the initially infected cells with uninfected cells four times. To investigate reactivation of persistent infection, CRFK cells chronically infected with FeFV were treated with trichostatin A (TA), a histone deacetylase inhibitor. TA induced higher FeFV production from the Coleman strain carrier culture and also induced marked syncytium formation. In contrast, human foamy virus, which contains less homologous long terminal repeat (LTR) and putative internal promoter (IP) sequences, persistently infecting baby hamster kidney cells was not reactivated by TA. The Sammy-1 strain of FeFV, from which a part of the U3 region in the LTR is naturally deleted, showed less reactivation. The Coleman LTR promoter-based ␤-Gal-expressing plasmid was activated in the persistently Coleman-infected cells in the presence of TA, whereas the Sammy-1 LTR was not activated. Furthermore, the amounts of Gag protein expressed did not change in the presence or absence of TA. Because the putative IP region was very similar between the two strains, the initiation by TA is relatively specific for LTR sequences, and, therefore, histone deacetylation is at least in part responsible for reactivation of FeFV from carrier cell culture. © 2001 Academic Press

INTRODUCTION (HFV) long terminal repeat (LTR) and internal promoter (IP), but the elements have little sequence identity (Kang Foamy viruses (FVs) have been isolated from a large number of mammalian species (Malmquist et al., 1969; et al., 1998). Tas transcription is initially induced by IP Riggs et al., 1969; Kennedy-Stoskopf et al., 1986; Achong (Yang et al., 1997). Once the level of Tas is upregulated, et al., 1971; Hooks and Gibbs, 1975; Hruska and Take- the IP is switched to using the LTR promoter for control- moto, 1975; Flanagan, 1992). The feline foamy virus ling transcription of the structural genes (Kang et al., (FeFV), which is classified as a member of the genus 1998). However, Tas is not found in viral particles. Thus, Spumavirus of family Retroviridae, was first isolated in after chronic FV infection, the reactivation mechanism is 1969 (Kasza et al., 1969); several strains of FeFV and still unknown. FeFV-related viruses have been reported since then Recent investigations have revealed that histone (Riggs et al., 1969; Mochizuki and Konishi, 1979; Ikeda et acetyltransferases are critical in activating and silencing al., 1997). Most of the FVs are highly cytopathogenic, promoter activities by altering the chromatin structure leading to cell death in vitro (Kasza et al., 1969; Mergia et (Pfeffer and Vidali, 1991). Trichostatin A (TA) is a specific al., 1996; Mikovits et al., 1996); however, paradoxically, inhibitor of histone deacetylases (Yoshida et al., 1990). TA persistently infected cells have been established in vitro has no effect on HIV-1 retroviral promoter activity when without cell death (Ikeda et al., 1997). The mechanisms of HIV promoter-reporter plasmids are unintegrated (Lint et persistent infection with FeFV are not yet understood. al., 1996). However, upon integration, TA activates HIV-1 For FV transcription, Bel-1 protein is known to be transcription (Sheridan et al., 1997) as well as human absolutely required as a transactivator (Tas), similar to papillomavirus promoter activity (Zhao et al., 1999) and Tat of HIV (Baunach et al., 1993). A putative Tas-specific expression of a transduced adenovirus promoter–lacZ binding site was found in both the human foamy virus gene (Chen et al., 1997). These reports suggest that TA is a useful tool for highly expressing some transduced genes. Early studies, in which FVs were applied as gene 1 To whom reprint requests should be addressed at the Department delivery vehicles (Bieniasz et al., 1997; Schmidt and of Veterinary Microbiology, Graduate School of Agricultural and Life Sciences, University of Tokyo, 113-8657 Tokyo, Japan. Fax: ϩ81-3-5841- Rethwilm, 1995; Russell and Miller, 1996), were carried 8184. E-mail: [email protected]. out despite the fact that the infectious virus titers were

TABLE 1 phological changes in S7801 and Sammy-1 carrier cul- Reactivation of FeFV Strains in CRFK Cells by Treatment with TA tures (data not shown). We also examined the effect of TA treatment by re- Persistent infection Acute infection verse transcriptase (RT) assays as described under Ma- terials and Methods. Virion-associated RT activity in the TA dilution Coleman S7801 Sammy-1 Coleman Coleman carrier culture supernatant increased from

Ϫ Ϯ Ϯ ␮ 10 4 4.5 Ϯ 0.3 1.5 Ϯ 0.1 1.3 Ϯ 0.3 NT 850 10 to 1433 21 dpm with the treatment of 3 M 10Ϫ5 2.3 Ϯ 0.2 1.0 Ϯ 0.2 1.2 Ϯ 0.3 NT TA, while RT activity of S7801 and Sammy-1 did not show 10Ϫ6 1.2 Ϯ 0.2 1.0 Ϯ 0.3 0.8 Ϯ 0.1 NT any significant increase (from 603 Ϯ 40 to 846 Ϯ 36 and Ϫ 10 7 1.2 Ϯ 0.4 1.0 Ϯ 0.3 0.7 Ϯ 0.2 NT from 520 Ϯ 26 to 487 Ϯ 30, respectively). As a negative Ϫ8 Ϯ Ϯ Ϯ 10 1.0 0.2 0.8 0.2 0.8 0.2 NT control, RT activity in the supernatant of CRFK culture 0 0.8 Ϯ 0.1 0.8 Ϯ 0.2 0.7 Ϯ 0.2 2.5 Ϯ 0.2 was 64 Ϯ 4 dpm. Note. The experiment was repeated three times. Values are repre- To compare the effect of other reagents on viral reac- Ϫ4 ϭ ␮ sented as mean virus titers by log10 TCID50/ml. 10 3 MTA tivation, we treated the cells with 5Ј-azacytidine, which concentration; NT, not tested. has been used to reactivate endogenous genes the promoters and enhancers of which are methylated (Fergu- son et al., 1995; Lee et al., 1995; Yoshiura et al., 1995). marginal (Schmidt and Rethwilm, 1995; Bieniasz et al., This reagent slightly reactivated viral production from all 1997); however, as yet there have been no studies of lines (Table 2). However, the reactivity is rather low whether TA regulates the FV promoter. compared with TA treatment of the coleman strain. In Because of the proposed use of FeFV vectors for gene contrast, treatment with sodium butyrate, which inhibits therapy (Bieniasz et al., 1997) and elucidation of initiation histone deacetylases and has a number of other activi- of FV infection (Yu et al., 1996), it is of interest to deter- ties (Kruh, 1982), did not reactivate viral production at a mine whether treatment with TA could be responsible for concentration of 50 mM (Table 2). Initial tests performed the enhancement of FeFV replication. In this study we with a broad range of sodium butyrate concentrations therefore examined whether TA can break the persistent yielded similar results (data not shown). These results state of FeFV-infected cells and also considered which suggest that the mechanism for reactivation of viral factors may be related to the chronicity of FeFV infection. transgenes may be different from the activation of some endogenous genes. Thus, histone acetylation is likely to RESULTS Reactivation of FeFV production from carrier cell culture by treatment with TA To determine whether the inhibition of histone deacetylation was the cause of reactivation of FeFV, we treated each of three FeFVs (Coleman, S7801, and Sammy-1 strains) and HFV carrier Crandell feline kidney (CRFK) and baby hamster kidney (BHK) cells, respectively, with titrated amounts of TA for 3 days (0.3 nM to 3 ␮M). Viral titers in the culture supernatant were deter- mined as 50% tissue culture infective dose (TCID50)by the cytopathic effect (CPE) assay. Although the viral titer was initially low in the supernatant of carrier culture, the TA treatment raised the viral production from the Coleman-carrying cells in a dose-dependent manner (Ta- ble 1). However, this reagent was not so effective against S7801, Sammy-1 (Table 1), and HFV (data not shown). Furthermore, after the treatment with TA, CRFK cells persistently infected with Coleman virus were stained with May–Gru¨nwald–Giemsa and were examined microscopically. The morphology of carrier cell cultures was dramatically changed by TA treatment. TA-treated FIG. 1. Morphological alterations of FeFV-infected CRFK cells caused Coleman carrier culture exhibited marked CPE with large by TA treatment. CRFK cells persistently infected with Coleman (C to F) or mock-infected (A and B) were treated with TA for 3 days at a syncytia (Figs. 1D, 1E, and 1F), very different from cul- concentration of 3 ␮M (B, D, and F) or 0.3 nM (E) or were left untreated tures not treated with TA (Fig. 1C) or control CRFK cells (A and C). After May–Gru¨nwald–Giemsa staining, cells were examined (Figs. 1A and 1B). However, we could not find any mor- microscopically. Magnifications: A to E, ϫ100; F, ϫ200. CHRONIC INFECTION OF FELINE FOAMY VIRUS 317

TABLE 2 Gag expression and morphology of viral structure Reactivation of FeFV in Persistently Infected Cells after Treatment in FeFV with 5-Azacytidine and Sodium Butyrate We examined viral protein expression in the carrier Virus strain 5-Azacytidine Butyrate None culture with or without TA treatment by immunoblotting (Fig. 3). The lysates of three FeFV carrier cells were Coleman 1.8 Ϯ 0.2 0.8 Ϯ 0.1 0.8 Ϯ 0.1 subjected to SDS–PAGE, transferred to membranes, and S7801 1.8 Ϯ 0.3 0.7 Ϯ 0.2 0.8 Ϯ 0.2 stained with FeFV-infected cat serum. An approximately Ϯ Ϯ Ϯ Sammy-1 1.5 0.2 0.8 0.2 0.7 0.2 53-kDa band corresponding to Gag was observed in all Note. The experiment was repeated three times. Values are pre- the carrier cells. The CRFK cells as a negative control did sented as mean virus titers by log10 TCID50/ml. Concentrations of not show any positive bands (Fig. 3). Unexpectedly, ex- 5-azacytidine and butyrate are 3 ␮M or 50 mM, respectively. pression levels of Gag proteins in the presence of TA (Fig. 3, lane 3 of Coleman, lane 5 of S7801, and lane 7 of Sammy-1) were the same as in its absence (Fig. 3, lane direct viral production in the initial phase of replication in 4 of Coleman, lane 6 of S7801, and lane 8 of Sammy-1). the chronically infected cells, but sensitivity to the acety- Bands of approximately 48 kDa, which were thought to lase reagent is different among the three viral strains. be a processed Gag protein, were also found in the Coleman and S7801 carrier cells, but not in Sammy-1 Partial deletion of U3 in the LTR sequence of FeFV (Fig. 3). Electron microscopy (EM) was used to examine the The proviral nucleotide sequence of the FeFV long budding of FeFV particles from the plasma membrane terminal repeat (LTR) region was analyzed and com- after treatment with TA. The numbers of budded pared among the three different FeFV strains (Coleman, Coleman and S7801 particles were increased to hun- S7801, and Sammy-1) aligned with the sequence of FUV dreds of virions per electron microscopic area by TA prototype strain (Fig. 2). The Coleman LTR was 1390 treatment, but this was not observed in cells infected nucleotides long and corresponds to the FUV LTR. Ho- with the Sammy-1 strain. Cells infected with Coleman mology of nucleic acid sequences between Coleman virus accumulated intracellular naked viral capsids (Fig. LTR and FUV and HFV LTRs was 97.7 and 47.1%, respec- 4). Interestingly, Coleman particles contained spike-like tively. On the other hand, S7801 and Sammy-1 LTRs were structures, probably representing envelope protein, different from the FUV LTR in length (1372 and 1371 whereas S7801 and Sammy-1 possessed multimem- nucleotides). Although the putative transcriptional factor brane-enclosed particles, with no spike appearance, binding motifs, such as heat-shock transcription factor containing an electron-dense viral core (Fig. 4). These and AP-1 transcription factor, are conserved in the LTR particles were not observed in the control CRFK cells promoter region, we found a deletion in the U3 region of (Fig. 4). S7801 and Sammy-1 (from nt 615 to nt 641) (Fig. 2). We also examined the nucleotide sequence of putative in- DISCUSSION ternal promoter regions among the three strains, but did not find any significant differences (data not shown). The In this study, we show that TA treatment induces viral HFV LTR did not contain homologous DNA sequences production at a higher titer from the carrier culture of corresponding to the deleted region in the U3 of S7801 FeFV/CRFK. Chen et al. (1997) have recently demon- and Sammy-1 LTRs. From these data, it is concluded that strated that the treatment with 5-azacytidine and sodium FeFV reactivation by TA treatment is LTR-specific and butyrate significantly increased reactivation of adenovi- dependent on this deleted promoter element. rus promoter–lacZ gene expression. In addition, both To investigate reactivity of Tas against the LTR pro- human and simian FVs have been reported to be reac- moter caused by TA treatment, ␤-galactosidase (␤-Gal) tivated by 5-azacytidine (Hoota and Loh, 1987; Schweizer assays were performed. Three LTR reporter plasmids et al., 1993), and TA reactivates HIV-1 transcription derived from Coleman, S7801, and Sammy-1 FeFV strains (Sheridan et al., 1997). Although butyrate showed less were transiently transfected into FeFV carrier cells. TA reactivation than TA, these data suggest that histone intensively induced high FeFV promoter activity of deacetylation is important for reactivation of FeFV. Coleman LTR–␤-gal reporter in Coleman carrier cultures. The Japanese isolates, S7801 and Sammy-1, contained In contrast, LTR of the other two strains did not (Table 3). deletions in the 5Ј-LTR promoter U3 regions compared S7801 and Sammy-1 LTRs were slightly activated by TA with the American isolate, Coleman (Fig. 2). HFV variants in their own virus carrier cultures (Table 3). Control pSV with the largest U3 deletions showed low ability to rep- promoters were not activated by Tas in the presence or licate in human cell cultures (Schmidt et al., 1997). In the absence of TA (Table 3). These data suggest that Tas addition, the nucleic acid sequences of the putative IP is crucial to viral transcription and that TA enhances the region and the U5 region of the FeFV and HFV LTRs were transactivation in an LTR-dependent manner. conserved (Kang et al., 1998), suggesting that deletion of 318 HATAMA ET AL.

FIG. 2. Genome organization and alignment of the nucleotide sequence of the promoter-enhancer region in FeFV. The complete 5Ј-LTR sequences among three FeFV strains (Coleman, S7801, and Sammy-1) are presented with the prototype FeFV FUV sequence. The scale at the top is drawn with respect to the start of the U3 region of the 5Ј-LTR. Numbers in the alignment of the sequences represent distances along the genome in base pairs. Deletions in the U3 promoter region in the 5Ј-LTR are boxed. The end of the U3, R, and U5 regions are represented by solid arrowheads with bars. Transcriptional factor binding motifs in the promoter region are marked as solid arrows. the U3 promoter region is related to the reactivation of view that the presence of Bel-1 is absolutely necessary FeFV in vitro. for LTR-mediated transcription. Interestingly, the level of The Bel-1 protein has been reported to be an essential expression of Gag was not altered by treatment with TA, requirement for LTR- and IP-mediated FV transcription and no difference in the amounts of Gag mRNA was (Baunach et al., 1993). Our results strongly support the detected by semiquantitative reverse transcriptase (RT)– CHRONIC INFECTION OF FELINE FOAMY VIRUS 319

FIG. 2—Continued

PCR (unpublished data). Further, in the presence or ab- tion (Saib et al., 1993). Histone deacetylation by TA is sence of TA, Hirt’s extracted DNA copies (Hirt, 1967) of likely to direct initiation of Bel-1 expression and LTR Coleman carrier cells were equivalent to those of S7801 deletion may be related to this initiation. Thus, initiation carrier cells. These data suggest that TA treatment has of FeFV reactivation by TA may involve the deleted ele- no effect on the accumulation of unintegrated FeFV DNA ment of the U3 promoter. Investigations of the precise and that TA could only change initiation of Bel-1. It has region spanning the putative regulator element are cur- been reported that Bel-1-defective forms of HFV provi- rently underway. ruses can prevent cell lysis and lead to persistent infec- In the case of the Sammy-1 strain, Gag protein did not 320 HATAMA ET AL.

TABLE 3 Activation of FeFV LTR Promoter-Based ␤-gal Reporter Plasmids on FeFV/CRFK Cells by Treatment with TA

␤-Gal-expressing plasmids

Transactivator Coleman LTR S7801 LTR Sammy-1 LTR pSV-␤gal

Coleman 104 Ϯ 6/68 Ϯ 10* 44 Ϯ 8/87 Ϯ 289Ϯ 5/80 Ϯ 4 0/0 S7801 56 Ϯ 2/35 Ϯ 8* 28 Ϯ 4/20 Ϯ 325Ϯ 3/29 Ϯ 3 0/0 Sammy-1 17 Ϯ 2/14 Ϯ 38Ϯ 1/14 Ϯ 115Ϯ 1/9 Ϯ 1 0/0

Note. The number of positive cells was counted by microscopic analysis after ␤-galactosidase staining. The sets of figures are TAϩ/TAϪ. * P Ͻ 0.01.

appear to be efficiently cleaved. Because it has been subcloned and used for CPE and ␤-Gal assays. CRFK suggested that processing of Gag has a critical role in cells persistently infected with FeFVs were established infecting host cells, infection of progeny virus to new as described previously (Miyazawa et al., 1995) and were cells in vitro may be related to Gag cleavage by protease. cultivated in Iscove’s modified Dulbecco’s medium Therefore, viral particles might have been rarely detected (Gibco. Grand Island, NY) supplemented with 10% heat- in persistently infected cells in EM analysis. Although inactivated fetal bovine serum and antibiotics. spike-less alteration of FV morphology has been observed in chimeric FVs (Pietschmann et al., 1999), our Viral titration results indicate the possibility of an alteration in the viral ␮ construct of Sammy-1. The morphological alteration is Twofold serial dilutions of the supernatant (100 l) likely to cause incomplete cleavage of the Gag protein from the infected cell culture were added to cells seeded and the uncleaved Gag may also contribute to the per- in a 96-well plate as described previously (Ikeda et al., sistent state via a lesser infectivity of viral particles; 1997). After cells were passaged two times at 6-day however, more precise examination needs to be done to intervals, infection by FeFV was judged by the CPE ob- define viral capsid construction. served by microscopy. The virus titers are presented at the 50% TCID50. MATERIALS AND METHODS RT assay Viruses and cells Virion-associated reverse transcriptase RT activity Three FeFV strains, American isolate Coleman (Ikeda was measured with 2.5 ␮Ci of [methyl-3H]deoxythymi- et al., 1997) and Japanese isolates S7801 (Mochizuki and dine 5Ј-triphosphate with 2 mM Mn2ϩ by the method of Konishi, 1979) and Sammy-1 (Miyazawa et al., 1995), Johnson et al. (Johnson et al., 1989). were obtained as described previously. The HFV strain, HS007 (Adachi et al., 1995), was a kind gift of Dr. A. Plasmid constructs and ␤-Gal assay Adachi. RT nonproducing cells derived from CRFK (ATCC CCL 94) (Crandell et al., 1973), designated NV112, were The LTR region was amplified from total RNA extracted from FeFV carrier cell cultures by PCR (primer F, TC- CAAGTGATGTTGCTTCCC; and primer R, CAGTAAGGTA- AGGTCCCTAG) using LA-Taq polymerase (Takara, Kyoto, Japan) and inserted into the pCR2.1 TA cloning vector (pCR/LTR1 of Coleman, pCR/LTR2 of S7801, and pCR/ LTR3 of Sammy-1). The DNA was automatically se- quenced by M13 primer using a DSQ-2000L machine (Shimazu, Tokyo, Japan). The sequences of Coleman, S7801, and Sammy-1 LTRs described here have been submitted to DNA Data Bank of Japan with the following FIG. 3. Detection of Gag-specific proteins in FeFV carrier cells. CRFK accession numbers: AB042566, AB042567, and cells persistently infected with Coleman (lanes 3 and 4), S7801 (lanes AB042568, respectively. The Accession No. of the FUV 5 and 6), or Sammy-1 (lanes 7 and 8) strains or mock-infected (lanes 1 LTR used here is Y08851. FeFV enhancer and promoter- and 2) were treated with TA or left untreated. After three days of ␤ incubation, cells were harvested, electrophoresed by SDS–PAGE, and directed -Gal expression plasmids (pSV/LTR-1, pSV/ analyzed by immunoblotting with FeFV Coleman-infected cat serum. LTR-2, pSV/LTR-3) were constructed by introducing the The molecular masses of proteins are given in the left margin. 1.4-kb EcoRI (blunted) fragment of pCR/LTR1-3 into pSV- CHRONIC INFECTION OF FELINE FOAMY VIRUS 321

FIG. 4. Electron micrographs of FeFV persistently infected CRFK cells. FeFV Coleman, S7801, and Sammy-1 carrier cells were treated with 50 ␮M of TA or were untreated. After 3 days, cells were fixed and then subjected to electron microscopy. Scale bar, 200 nm (FeFV-infected cells) and 1 ␮m (Control).

␤gal cut by HindIII (blunted). All plasmids were prepared Immunoblotting for ␤-Gal assays by alkaline lysis and purification by Electrophoresis was performed in 10% polyacrylamide cesium chloride centrifugation. Transient transfection gels containing 0.1% SDS. After transfer to polyvinylidene into the FeFV carrier cells was performed using the difluoride membranes (Millipore, MA), the blotted pro- lipofectin liposomal transfection reagent as specified by teins were blocked with 5% dry milk in PBS with 0.1% the manufacturer (Life Technologies, Germany). Briefly, NP-40 overnight. Anti-FeFV and control feline sera ob- subconfluent cells in 60-mm-diameter plates were trans- tained from FeFV-infected and specific-pathogen-free fected with 5 ␮g of total plasmid DNA and 25 ␮lof cats were used as a first antibody, and anti-cat IgG lipofectin (GIBCO) as described previously (Fujii et al., conjugated to the alkaline phosphatase (Boehringer- 1996). For ␤-Gal assays, cells were fixed in 0.5% glutar- Mannheim, Germany) was used as the secondary anti- aldehyde and stained with 2% X-Gal (Takara) substrate body. The membrane was stained as described previ- with 50 mM potassium ferri/ferrocyanide buffer. Specific ously (Otake et al., 1994). activation of LTR was calculated as follows; specific activity of ␤-Gal ϭ positive cell numbers of experimental Electron microscopic analysis sample Ϫ the spontaneously positive cell number of the control sample. Spontaneous activation was measured FeFV-infected and uninfected cells were washed by transfection of LTR–␤-Gal plasmids alone into unin- and fixed in 2% glutaraldehyde followed by 0.1 M fected CRFK cells. The values shown are the means Ϯ phosphate buffer (pH 7.2) containing 2% osmium tet- SD of at least three independent transfections. roxide. Samples were then stained with 1% uranyl Profiles of transcription factor binding sites were an- acetate, dehydrated with a graded series of ethanol alyzed by TFSEARCH ver. 1.3 in the GenomeNet web site. dilutions followed by 100% ethanol propylene oxide, 322 HATAMA ET AL. and embedded in epoxy resin. Thin sections were Kang, Y., Blair, W. S., and Cullen, B. R. (1998). Identification and func- cut and stained with lead citrate. The sections tional characterization of a high-affinity Bel-1 DNA binding site lo- cated in the human foamy virus internal promoter. J. Virol. 72, 504– were viewed and photographed using a JEOL Model 511. 1200 EX. Kasza, L., Hayward, A. H., and Betts, A. O. (1969). Isolation of a virus from a cat sarcoma in an established canine melanoma cell line. Res. Vet. Sci. 10, 216–218. ACKNOWLEDGMENTS Kennedy-Stoskopf, S., Kennedy-Stoskopf, M., Eckhaus, M. A., and Standberg, J. D. (1986). Isolation of a retrovirus and a herpesvirus We thank Dr. T. 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