RNA from unique and repetitive DNA sequences ofHerpesvirus saimiri

VIROLOGY 100, 204-207 (1980)

RNA from Unique and Repetitive DNA Sequences of Herpesvirus s a i m i r i 1 SHARON TRACY AND RONALD C. DESROSIERS 2 New England Regional Primate Research Center, Harvard Medical School, 1 Pine Hill Drive, Southborough, Massachusetts 01772 Accepted October 2, 1979 Herpesvirus saimiri DNA contains repetitive DNA at its termini that comprises about 30% of the virion DNA. After labeling late in infection, cytoplasmic RNA derived from these repetitive sequences was found to be below our limits of detection. Less than 1%, if any, of the viral-specific cytoplasmic poly(A)-containing RNAs contain sequences hybridizable to the isolated repetitive DNA.

Herpesvirus saimiri (H. saimiri) causes no apparent disease in its natural host, squirrel monkey (Saimiri sciureus), and can be readily isolated from lymphocytes of most squirrel monkeys. H. saimiri, however, produces rapidly progressing lymphomas and subsequent death in marmosets and owl monkeys (1). The genome of infecious H. saimiri has been termed M-DNA since it consists of heavy (H-high GC) and light (L-low GC) regions of DNA covalently linked. A unique region of 70 × 106 daltons (36% G + C, L-DNA) is flanked by a variable number of repetitive units of H-DNA (71% G + C) at each terminus. Each repeat unit of H-DNA is 830,000 daltons and H-DNA comprises about 30% (30 × 106 daltons) of M-genomes (2). Defective virus particles are also found in lower amounts (-10%) in virus preparations and these contain long H-DNA molecules (2). We have begun our studies of the transcription of the H. saimiri genome by analyzing the relative transcription from Hand L-DNA sequences late after permissive infection of owl monkey kidney (OMK) cells. The H-DNA repeat unit (830,000 daltons) is large enough to code for an average size mRNA and polypeptide. Also, since 1This work was supported by Grant No. RR00168-17, Animal Resources Branch, National Institutes of Health and Contract N01 CP 81005 from the Virus Cancer Program of the National Cancer Institute. 2 To whom requests for reprints should be addressed. 0042-6822/80/010204-04502.00/0 Copyright© 1980by AcademicPress. Inc. All rightsof reproductionin any formreserved.

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other viral mRNAs are frequently generated by splicing from noncontiguous sequences, it seemed possible to us that many cytoplasmic mRNAs could contain sequences transcribed from H-DNA at their 5' end. Permissive OMK cells were labeled late after infection with [3H]uridine and [3H]RNA was isolated from the cytoplasm. The cytoplasmic RNA was further separated into RNA containing (poly(A+)) or lacking (poly(A-)) terminal poly(A) by two cycles of oligo(dT)-cellulose chromatography. After sedimentation through sucrose gradients the 3H-poly(A+)RNA produced a broad heterogeneous profile with the peak of radioactivity around 18 S (data not shown). This is expected for a heterogeneous population of mRNA molecules and is similar to what has been seen previously for a complex population of cellular or viral mRNA molecules (3, 4). To confirm that the isolated M-, L-, and H-DNAs functioned as expected in the hybridizations employed here, denatured [3H]M-DNA was hybridized to the isolated M-, L-, and H-DNAs immobilized on nitrocellulose filters. As expected, [3H]M-DNA hybridized to M-DNA greater than 90% (Fig. 1A). [3H]M-DNA also hybridized well with isolated L-DNA (Fig. 1B) and H-DNA (Fig. 1C). The maximal hybridization of 22% with H-DNA in Fig. 1 is close to what is expected for [~H]TTP-labeled M-DNA. The analysis of the [3H]M-DNA by CsC1 equilibrium centrifugation after shearing also indicated approximately 20% of the label

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FIG. 1. OMK cells were infected with H. saimiri strain 11 (m.o.i. ~ 0.5) 2 days following a 1:3 split. Four days after infection the medium (Eagle's MEM, Gibco F l l ) was removed and replaced with medium containing 25 t~Ci/ml [5-3H]uridine (28 Ci/mmol, New England Nuclear). After 18 hr of labeling, the cells were scraped from the plate, washed once with phosphate buffered saline, and cytoplasmic RNA was then prepared as previously described (4). Poly(A+) and p o l y ( A - ) R N A were separated by chromatography on oligo(dT)-cellulose (collaborative research T2) using 0.3 M NaC1 in the high salt buffer. M-DNA (1.705 g/ml) was prepared from H. saimiri No. 11 virions by equilibrium centrifugation in CsC1 as previously described (2). High molecular weight H-DNA (1.730 g/ml) was also purified from virions by the same procedure except that a second CsC1 equilibrium centrifugation was employed to remove any detectable M-DNA contamination. L-DNA was prepared following cleavage of M-DNA with restriction endonuclease SmaI; S m a I cleaves four times within each H-DNA repeat unit but it does not cleave within L-DNA (2). Sinai cleaved M-DNA was sedimented in a 10-30% sucrose gradient containing 1 M NaC1, 0.05 M Tris-HC1 pH 7.5, and 0.001 M EDTA: fractions containing L-DNA around 40 S were pooled and the L-DNA was recovered by precipitation with ethanol. For use in control hybridizations (A, B, and C), M-DNA was labeled in vitro with [3H]TTP to a specific activity of 1.3 x 107 cpm/teg of DNA by the nick repair method (8). [3H]RNA or denatured [ZH]DNA was hybridized to various amounts of unlabeled DNA immobilized on filters. DNA, denatured in a small volume with NaOH and then neutralized (technique 2 of Ref. (9)), was bound to 13 ram, 0.45-tem nitrocellulose filters (Schleicher and Schuell BA85) in 6 x SSC, rinsed with 6 x SSC, and baked at 80° for 2 hr. The hybridization solution contained [3H]RNA or denatured [3H]DNA as indicated in 4 x SSC, 0.1% SDS in a total volume of 300 t~l. Hybridization was performed at 67° for 20 hr in Kimble 7 ml scintillation mini-vials. Following hybridization the liquid was removed and each filter washed three times with 2 x SSC (5-min incubation for each wash). Each filter was then incubated with 0.5 ml 2 x SSC for 1 hr at room temperature. After removing the 2 x SSC, each filter was then incubated with 2 x SSC, 0.1% SDS, for 30 min at 60°. The liquid was removed, the filter cooled and washed a final three times with 2 x SSC (5 min each). Each filter was dried, cooled, and counted with PPO and POPOP in toluene. In A, B, and C constant amounts of denatured [3H]M-DNA were hybridized with M-, L-, or H-DNA, respectively, immobilized on filters. In D, constant amounts of 3H-poly(A+)RNA were hybridized with various DNAs immobilized on filters. (D) ( I ) L-DNA immobilized on filters; (O) M-DNA immobilized on filters; ( e ) DNA from herpes simplex virus type 1 grown in OMK cells immobilized on the filter; (×) H-DNA immobilized on filters.

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in the H-DNA sequences. Hybridization of [3H]L-DNA with H-DNA immobilized on filters and [3H]H-DNA with L-DNA immobilized on filters indicated little (less than 5%) cross contamination (data not shown). Hybridization of 3H-poly(A+)RNA from infected cells to M-DNA immobilized on filters revealed greater than 50% hybridization (Fig. 1D). This indicates that most of the mRNA synthesis in the cell at the time of labeling was devoted to viral mRNA synthesis. Hybridization of the ~H-poly(A+)RNA to isolated L-DNA was sufficient to account for all the viral-specific poly(A+)RNA (Fig. 1D). Hybridization of the 3H-poly(A+)RNA to H-DNA was below detectable levels (less than 1%) (Fig. 1D). The use of twice purified H-DNA was important for lowering the limit of detectability to this level. The ~H-poly(A+)RNA hybridizing to L- or M-DNA was greater than 90% resistant to digestion by 20 t~g/ml pancreatic RNase A for I hr at room temperature while unhybridized 3H-poly(A+)RNA was completely sensitive (data not shown). The amounts of 3H-labeled viral-specific RNA in each fraction in a typical experiment are shown in Table 1. Use of this filter hybridization technique has the advantage of detecting mRNA molecules with only a short stretch of nucleotides hybridizable to H-DNA present as a "leader sequence" even if these short stretches comprise less than a few percent of the total sequence. In the absence of pancreatic RNase A, a short stretch of hybridized [3H]RNA would retain 3H sequences distal to it on the filter even if these distal sequence were not hybridizable. Control experiments have shown that 3H-

poly(A+)RNA taken through this hybridization procedure are at least 50% of the original mean size. We thus feel that less than 1% of the viral mRNA molecules present late in infection contain even a short stretch of sequence transcribed from the H-DNA region. This conclusion is limited only by the limit lengths of RNA that can hybridize under these conditions. A short stretch of eight nucleotides, for example, from the H region would probably not be detected. Thermal stability as a function of oligonucleotide length has been described (10). Quantitative evaluation of viral RNA sequences in the poly(A-)RNA fraction was hampered by the fact that less than 0.5% of the 3H-poly(A-)RNA was viral specific. Thus less than 25% of all the viralspecific [3H]RNA sequences were in the poly(A-)RNA fraction and no specific hybridization to HoDNA was observed. In conclusion, although repetitive H-DNA sequences represent 30% of the H. saimiri genome, less than 1%, if any, of the mRNA present late in infection contain sequences from this region of the genome. It is interesting to note that lymphotropic herpesviruses of other New World primates related to H. saimiri exhibit considerably greater divergence in the H-DNA sequences than in the L-DNA sequences (5, 6). If H-DNA is not involved in coding for an essential protein, perhaps it can tolerate greater base sequence divergence. Future transcription studies from this laboratory will concentrate on RNA made from a 1.1 × 106-dalton region of the genome known to be deleted in attenuated strains of H. saimiri (7) (Koomey, et al., to be published).

TABLE 1 MEASUREMENT OF VIRAL-SPECIFIC RNA

Poly(A+)RNA Poly(A-)RNA

Total cpm recovered

cpm hybridizable to MoDNA

cpm hybridizable to L-DNA

cpm hybridizable to H-DNA

2,800,000 94,000,000

2,100,000 a <470,000

2,100,000 a

<21,000

These values were estimated from additional hybridizations to those shown in Fig. 1D.

SHORT COMMUNICATIONS REFERENCES 1. DANIEL, M. D., MELENDEZ, L. V., HUNT, R. D., and TRUM, B. F., In "Pathology of Simian Primates" (R. N. T.-W. Fiennes, ed.), Pt. II, pp. 592-611. Karger, Basel, 1972. 2. BORNKAMM,G., DELIUS, H., FLECKENSTEIN,B., WERNER, F.-J., and MULDER, C., J. Yirol. 19, 154-161 (1976). 3. STRINGER, J., HOLLAND, L., SWANSTROM, R., PIVO, K., and WAGNER, E., J. Yirol. 21, 889-901 (1977). 4. DESROSIERS, R., FRIDERICI, K., and ROTTMAN, F., Proc. Nat. Acad. Sci. USA 71, 3971-3975 (1974). 5. MULDER,C., FLENKENSTEIN,B., DANIEL,M. D.,

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and SHELDRICK, P., In "Abstracts of the Fourth International Congress for Virology," p. 524, Center for Agricultural Publishing and Documentation, The Hague, 1978. FLECKENSTEIN,B., BORNKAMM,G., MULDER, C., WERNER, F.-J., DANIEL,M. D., FALK, L., and DELIUS, H., J. Virol. 25, 361-373 (1978). SCHAFFER, P., FALK, L., and DEINHARDT, F., J. Nat. Cancer Inst. 55, 1243-1246 (1975). DESROSIERS,R., MULDER,C., and FLECKENSTEIN, B., Proc. Nat. Acad. Sci. USA 76, 3839-3843 (1979). FLECKENSTEIN, B., MULLER, I., and WERNER, F.-J., Int. J. Cancer 19, 546-554 (1977). GRALLA, J., and CROTHERS, D. M., J. Mol. Biol. 73, 497-511 (1973).