JOURNAL OF VIROLOGY, May 1977, p. 577-582 Copyright © 1977 American Society for Microbiology
Vol. 22, No. 2 Printed in U.S.A.
Adenovirus Type 2 Nuclear RNA Accumulating During Productive Infection STEVEN L. BACHENHEIMERI The Rockefeller University, New York, New York 10021 Received for publication 22 September 1976
The viral-specific nuclear RNA which accumulates early and late during productive infection of HeLa cells by adenovirus type 2 (Ad2) has been characterized with respect to its size and stability after denaturation by Me2SO. Early nuclear transcripts, under nondenaturing conditions, sediment in the range 28 to 45S, but treatment with Me2SO prior to sedimentation results in a shift to about 20S. Late nuclear RNA accumulates as a composite of two populations of molecules: one with a broad size distribution centering on 45S under nondenaturing conditions and 18S) 1 Present address: Department of Bacteriology and free of degraded DNA (as judged by either abImmunology, The University of North Carolina, Chapel sorbance at 260 nm, or KOH-resistant, trichloroacetic acid-precipitable counts per minute) Hill, NC 27514. 577
578
J. VIROL.
NOTES
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0-
0U
0
1
X~~~~~~~~~ a.3 0 ___
t
that was synthesized and accumulated in the presence (25 ,ug/ml; closed circles, Fig. 2) or absence (open circles, Fig. 2) of cycloheximide (Fig. 2B and D), except for the tendency to accumulate slightly more higher-molecularweight (>32S) RNA as compared to RNA -20S in size in the absence of the drug under nondenaturing conditions. Therefore, it seemed justified to use cycloheximide in the preparation of early viral RNA to improve the efficiency of hybridization of Ad2 DNA. An analysis of the size and stability of accumulating viral-specific nuclear molecules is also presented in Fig. 2. Panels B and D represent sedimentation of viral RNA under nondenaturing and denaturing conditions, respec-
tively. In 50 mM NaCl, the bulk of viral RNA sedimented broadly between 28 and 45S, (fractions 30 to 14), with small amounts trailing down to 80 to lOOS in size. However, after denaturation of RNA with Me2SO, and in contrast to total nuclear RNA, there was a large shift in the size of viral RNA, to approximately 18 to 20S (fracSEDIMENTATION tions 32 to 22), with a slight shoulder in the FIG. 1. Susceptibility of reovirus RNA to RNase of 32S (fractions 20 to 22) and diminregion digestion before and after denaturation with Me2SO. Aliquots of 32P-labeled reovirus double-stranded ished amounts of RNA in the region greater RNA were either diluted into ETS buffer (10 mM than 45S RNA. Centrifugation of cytoplasmic EDTA, 10 mM Tris [pH 7.4], 0.2% SDS) or de- RNA under identical conditions in parallel natured with Me$SO as described in the legend to with nuclear RNA have shown that the size Fig. 2 and then layered on 10 to 30% sucrose-0.05 M distribution of most of the Me2SO-denatured, NETS (50 mM NaCi, 10 mM EDTA, and 10 mM nuclear virus-specific RNA is quite similar, if Tris [pH 7.4], and 02% SDS) gradients and cen- not identical, to that of cytoplasmic viral trifuged for 15 h at 30,000 rpm in the SW41 rotor. mRNA (data not shown). Similar observations One half of each fraction was directly precipitated with trichloroacetic acid. The other half was made have recently been made by Craig and Raskas 1 M in KCI and incubated at 40C for 30 min to pre- (3). The size distribution of total and viral-specipitate the SDS. NaCl was then added to 0.3 M, RNase A was added to 5 pg/ml, and the fraction was cific nuclear RNA synthesized late during inincubated at room temperature for 30 min. EDTA to fection is shown in Fig. 3. As was the case early 5 mM and 100 pg ofyeast RNA were added, and the during infection, the total RNA which had been samples were precipitated with trichloroacetic acid. labeled late in infection (panel A) was quite (A) Without MeSO. (B) With MesO. Symbols: *, stable to the action of Me2SO. Though the without RNase; O., with RNase. majority of incorporation of label was still into 45 and 32S preribosomal RNA (panel A), two were pooled and divided for centrifugation comments should be made. First, the amount of under identical conditions with and without label in 32S relative to 45S RNA has declined denaturation by Me2SO. significantly (down one-third compared with Early in infection (2 to 5 h postinfection), early patterns, data not shown), suggestive of a virtually all the detectable incorporation of iso- slower processing of the 45S precursor. Second, tope was into preribosomal 45 and 32S RNA as a result of both the overall decline in RNA (whose positions were not resolved in this ex- synthesis (18) and the significant incorporation periment). Cycloheximide appeared to have no of label into viral RNA (16), the labeled RNA effect on the size or stability of total nuclear sedimenting faster than the 45S RNA, characRNA (closed circles, Fig. 2), since total RNA is teristic of hnRNA, is more apparent. Panel B shown to be quite stable to the action of Me2SO. represents the corresponding size distributions Analysis of viral-specific RNA was carried of virus-specific RNA under nondenaturing out by hybridization of fractions from the su- conditions or after Me2SO denaturation. Before crose gradients to Ad2 DNA. This analysis denaturation, viral RNA was primarily found (Fig. 2) revealed little difference between RNA in size classes ranging from about 8,000 to 10
VOL. '22, 1977
NOTES
180
~~~~~~.D
nab
CLA .
0~~~~
',bs 6
c
A
10
20
30
10
20
30
Fraction number
FIG. 2. Size distribution of virus-specific nuclear RNA accumulating early during infection. HeLa cells (2 x 108) maintained in Joklik modified minimal essential medium (MEM) plus 5% fetal calf serum at 2 x 105 to 5 x 105/ml were concentrated to 107/ml and exposed to 2 x 103 viral particles per cell. After 30 min one halfof the culture was diluted to 10. cellslml in medium containing 25 Pg of cycloheximide for 1 h. The other half was washed three times with POf-free MEM and resuspended at 106 cell/ml in POrfree MEM plus 5% dialyzed fetal calf serum for 1 h. Infected cells treated with cycloheximide were labeled for 3.5 h with 5 mCi of [3H]uridine (30 Ci/mmol), and the untreated cells were labeled with 5 mCi of 32PO4. After harvesting, the cells were washed with phosphate-buffered saline and resuspended at 10' cellslml in RSB (10 mM NaCl, 10 mM Tris [pH 7.4], 1.5 mM MgCl [13D). After 5 min, cells were disrupted by Dounce homogenization, and the nuclei were recovered by sedimentation. Nuclei were washed once with RSB, resuspended in RSB, and detergent washed (14). After resedimentation the nuclei were disrupted, and RNA was purified as described previously (19). The purified RNA was resuspended in 0.05 M NETS (50 mM NaCl, 10 mM EDTA, 10 mM Tris [pH 7.4], 02% SDS) and centrifuged for 16 h at 16,000 rpm in the SW27 rotor through 15 to 30% sucrose-0.05 MNETS gradients. RNA sedimenting at or greater than 18S (as judged by absorbance at 260 nm) was pooled and precipitated with 2 volumes of EtOH. One half of the RNA was resuspended in 0.05 M NETS, and the other half was denatured with MeSO by resuspending in 1 volume of ETS (10 mM EDTA, 10 mM Tris [pH 7.4], 0.2% SDS) and incubating for 5 min at 370C, followed by addition of volume of dimethyl formamide and 9 volumes of MeSO buffered with 10 mM Tris (pH 7.4) and 10 mM EDTA, with 5-min incubations at 370C after each addition. Finally, the sample was diluted 1:3 with ETS. The RNA samples were layered on sucrose gradients and centrifuged for 16 h at 16,000 rpm in the SW27 rotor, collected
579
30,000 nucleotides, with a large peak around 12,000 nucleotides (35 to 405). After denaturation in Me2SO and sedimentation in a low-salt sucrose gradient, viral RNA was still present, but in decreased amounts, in the very large size range; the main peak was still present at 10,000 to 12,000 nucleotides, and approximately 50% of the viral RNA was present in molecules less than 8,000 nucleotides in length (compared with 25% under nondenaturing conditions). These results contrast those found early during infection, which showed that the bulk of viral sequences were in molecules less than 10,000 nucleotides long and that virtually all the large virus-specific RNA was unstable to the action of Me2SO (Fig. 2B and D). The 35 to 40S viral RNA accumulating late in infection and stable to Me2SO was further analyzed by hybridization to restriction enzymegenerated fragments of viral DNA. The Ad2 genome contains 6 EcoR-RI (15) and 11 major SmaI (C. Mulder, personal communication) fragments and can be illustrated as shown in Fig. 4, where the letters A through F and A through K represent EcoRI and SnaI fragments, respectively, of decreasing size and the numbers represent their position expressed as a percentage of the total genome length. Analyses of native and denatured RNA from the sucrose gradients represented in Fig. 3 revealed a broad size distribution of RNA complimentary of fragments EcoRI-B through F (data not shown). In contrast to these results, the size distribution of EcoRI-A-specific RNA appeared to be a composite of both a broad distribution and a peak around 35 to 40S (Fig. 5). Furthermore, while the broad component was relatively sensitive to the action of MeSO (a decrease in RNA >45S with a concomitant increase in RNA
Vol. 22, No. 2 Printed in U.S.A.
Adenovirus Type 2 Nuclear RNA Accumulating During Productive Infection STEVEN L. BACHENHEIMERI The Rockefeller University, New York, New York 10021 Received for publication 22 September 1976
The viral-specific nuclear RNA which accumulates early and late during productive infection of HeLa cells by adenovirus type 2 (Ad2) has been characterized with respect to its size and stability after denaturation by Me2SO. Early nuclear transcripts, under nondenaturing conditions, sediment in the range 28 to 45S, but treatment with Me2SO prior to sedimentation results in a shift to about 20S. Late nuclear RNA accumulates as a composite of two populations of molecules: one with a broad size distribution centering on 45S under nondenaturing conditions and 18S) 1 Present address: Department of Bacteriology and free of degraded DNA (as judged by either abImmunology, The University of North Carolina, Chapel sorbance at 260 nm, or KOH-resistant, trichloroacetic acid-precipitable counts per minute) Hill, NC 27514. 577
578
J. VIROL.
NOTES
a.
0-
0U
0
1
X~~~~~~~~~ a.3 0 ___
t
that was synthesized and accumulated in the presence (25 ,ug/ml; closed circles, Fig. 2) or absence (open circles, Fig. 2) of cycloheximide (Fig. 2B and D), except for the tendency to accumulate slightly more higher-molecularweight (>32S) RNA as compared to RNA -20S in size in the absence of the drug under nondenaturing conditions. Therefore, it seemed justified to use cycloheximide in the preparation of early viral RNA to improve the efficiency of hybridization of Ad2 DNA. An analysis of the size and stability of accumulating viral-specific nuclear molecules is also presented in Fig. 2. Panels B and D represent sedimentation of viral RNA under nondenaturing and denaturing conditions, respec-
tively. In 50 mM NaCl, the bulk of viral RNA sedimented broadly between 28 and 45S, (fractions 30 to 14), with small amounts trailing down to 80 to lOOS in size. However, after denaturation of RNA with Me2SO, and in contrast to total nuclear RNA, there was a large shift in the size of viral RNA, to approximately 18 to 20S (fracSEDIMENTATION tions 32 to 22), with a slight shoulder in the FIG. 1. Susceptibility of reovirus RNA to RNase of 32S (fractions 20 to 22) and diminregion digestion before and after denaturation with Me2SO. Aliquots of 32P-labeled reovirus double-stranded ished amounts of RNA in the region greater RNA were either diluted into ETS buffer (10 mM than 45S RNA. Centrifugation of cytoplasmic EDTA, 10 mM Tris [pH 7.4], 0.2% SDS) or de- RNA under identical conditions in parallel natured with Me$SO as described in the legend to with nuclear RNA have shown that the size Fig. 2 and then layered on 10 to 30% sucrose-0.05 M distribution of most of the Me2SO-denatured, NETS (50 mM NaCi, 10 mM EDTA, and 10 mM nuclear virus-specific RNA is quite similar, if Tris [pH 7.4], and 02% SDS) gradients and cen- not identical, to that of cytoplasmic viral trifuged for 15 h at 30,000 rpm in the SW41 rotor. mRNA (data not shown). Similar observations One half of each fraction was directly precipitated with trichloroacetic acid. The other half was made have recently been made by Craig and Raskas 1 M in KCI and incubated at 40C for 30 min to pre- (3). The size distribution of total and viral-specipitate the SDS. NaCl was then added to 0.3 M, RNase A was added to 5 pg/ml, and the fraction was cific nuclear RNA synthesized late during inincubated at room temperature for 30 min. EDTA to fection is shown in Fig. 3. As was the case early 5 mM and 100 pg ofyeast RNA were added, and the during infection, the total RNA which had been samples were precipitated with trichloroacetic acid. labeled late in infection (panel A) was quite (A) Without MeSO. (B) With MesO. Symbols: *, stable to the action of Me2SO. Though the without RNase; O., with RNase. majority of incorporation of label was still into 45 and 32S preribosomal RNA (panel A), two were pooled and divided for centrifugation comments should be made. First, the amount of under identical conditions with and without label in 32S relative to 45S RNA has declined denaturation by Me2SO. significantly (down one-third compared with Early in infection (2 to 5 h postinfection), early patterns, data not shown), suggestive of a virtually all the detectable incorporation of iso- slower processing of the 45S precursor. Second, tope was into preribosomal 45 and 32S RNA as a result of both the overall decline in RNA (whose positions were not resolved in this ex- synthesis (18) and the significant incorporation periment). Cycloheximide appeared to have no of label into viral RNA (16), the labeled RNA effect on the size or stability of total nuclear sedimenting faster than the 45S RNA, characRNA (closed circles, Fig. 2), since total RNA is teristic of hnRNA, is more apparent. Panel B shown to be quite stable to the action of Me2SO. represents the corresponding size distributions Analysis of viral-specific RNA was carried of virus-specific RNA under nondenaturing out by hybridization of fractions from the su- conditions or after Me2SO denaturation. Before crose gradients to Ad2 DNA. This analysis denaturation, viral RNA was primarily found (Fig. 2) revealed little difference between RNA in size classes ranging from about 8,000 to 10
VOL. '22, 1977
NOTES
180
~~~~~~.D
nab
CLA .
0~~~~
',bs 6
c
A
10
20
30
10
20
30
Fraction number
FIG. 2. Size distribution of virus-specific nuclear RNA accumulating early during infection. HeLa cells (2 x 108) maintained in Joklik modified minimal essential medium (MEM) plus 5% fetal calf serum at 2 x 105 to 5 x 105/ml were concentrated to 107/ml and exposed to 2 x 103 viral particles per cell. After 30 min one halfof the culture was diluted to 10. cellslml in medium containing 25 Pg of cycloheximide for 1 h. The other half was washed three times with POf-free MEM and resuspended at 106 cell/ml in POrfree MEM plus 5% dialyzed fetal calf serum for 1 h. Infected cells treated with cycloheximide were labeled for 3.5 h with 5 mCi of [3H]uridine (30 Ci/mmol), and the untreated cells were labeled with 5 mCi of 32PO4. After harvesting, the cells were washed with phosphate-buffered saline and resuspended at 10' cellslml in RSB (10 mM NaCl, 10 mM Tris [pH 7.4], 1.5 mM MgCl [13D). After 5 min, cells were disrupted by Dounce homogenization, and the nuclei were recovered by sedimentation. Nuclei were washed once with RSB, resuspended in RSB, and detergent washed (14). After resedimentation the nuclei were disrupted, and RNA was purified as described previously (19). The purified RNA was resuspended in 0.05 M NETS (50 mM NaCl, 10 mM EDTA, 10 mM Tris [pH 7.4], 02% SDS) and centrifuged for 16 h at 16,000 rpm in the SW27 rotor through 15 to 30% sucrose-0.05 MNETS gradients. RNA sedimenting at or greater than 18S (as judged by absorbance at 260 nm) was pooled and precipitated with 2 volumes of EtOH. One half of the RNA was resuspended in 0.05 M NETS, and the other half was denatured with MeSO by resuspending in 1 volume of ETS (10 mM EDTA, 10 mM Tris [pH 7.4], 0.2% SDS) and incubating for 5 min at 370C, followed by addition of volume of dimethyl formamide and 9 volumes of MeSO buffered with 10 mM Tris (pH 7.4) and 10 mM EDTA, with 5-min incubations at 370C after each addition. Finally, the sample was diluted 1:3 with ETS. The RNA samples were layered on sucrose gradients and centrifuged for 16 h at 16,000 rpm in the SW27 rotor, collected
579
30,000 nucleotides, with a large peak around 12,000 nucleotides (35 to 405). After denaturation in Me2SO and sedimentation in a low-salt sucrose gradient, viral RNA was still present, but in decreased amounts, in the very large size range; the main peak was still present at 10,000 to 12,000 nucleotides, and approximately 50% of the viral RNA was present in molecules less than 8,000 nucleotides in length (compared with 25% under nondenaturing conditions). These results contrast those found early during infection, which showed that the bulk of viral sequences were in molecules less than 10,000 nucleotides long and that virtually all the large virus-specific RNA was unstable to the action of Me2SO (Fig. 2B and D). The 35 to 40S viral RNA accumulating late in infection and stable to Me2SO was further analyzed by hybridization to restriction enzymegenerated fragments of viral DNA. The Ad2 genome contains 6 EcoR-RI (15) and 11 major SmaI (C. Mulder, personal communication) fragments and can be illustrated as shown in Fig. 4, where the letters A through F and A through K represent EcoRI and SnaI fragments, respectively, of decreasing size and the numbers represent their position expressed as a percentage of the total genome length. Analyses of native and denatured RNA from the sucrose gradients represented in Fig. 3 revealed a broad size distribution of RNA complimentary of fragments EcoRI-B through F (data not shown). In contrast to these results, the size distribution of EcoRI-A-specific RNA appeared to be a composite of both a broad distribution and a peak around 35 to 40S (Fig. 5). Furthermore, while the broad component was relatively sensitive to the action of MeSO (a decrease in RNA >45S with a concomitant increase in RNA
