JOURNAL OF VIROLoGY, Aug. 1976, p. 743-745 Copyright © 1976 American Society for Microbiology
Vol. 19, No. 2 Printed in U.S.A.
NOTES Polyadenylic Acid in the Genomic RNA of Mengovirus MILTON V. MARSHALL AND RALPH B. ARLINGHAUS* Departments of Biology* and Biochemistry, The University of Texas System Cancer Center, M. D. Anderson Hospital and Tumor Institute, Houston, Texas 77030 Received for publication 27 February 1976
The polyadenylic acid contained in 35S mengovirus RNA produced in infected BHK-21 cells contained approximately 94% AMP and was estimated to contain an average of 50 to 55 nucleotides. The polyadenylic acid is placed at the 3'-end of the genomic RNA based on the presence of significant levels of [3H]adenosine in complete alkali or RNase T2 digests of polyadenylic acid from [3H]adenosinelabeled 35S viral RNA.
Polyadenylic acid [poly(A)] sequences have been found in picornavirus RNA genomes such as poliovirus (3, 14, 16), encephalomyocarditis virus (6), Columbia S-K virus (7), mengovirus (9, 13), foot-and-mouth disease virus (N. K. Chatteijee, H. L. Bachrach, and J. Polatnick, Virology, in press), and rhinovirus (10). These poly(A) sequences range in size from 50 to 100 nucleotides in length. Some controversy exists with respect to the size of the poly(A) sequence in mengovirus RNA. Miller and Plagemann (9) have reported that mengovirus RNA produced in Novikoff rat hepatoma cells contained a short tract of poly(A) (15 to 17 residues). In contrast to this, Spector and Baltimore (13) showed that mengovirus RNA produced in L cells and HeLa cells contained a longer poly(A) sequence (50 to 70 residues). In the work described here, mengovirus RNA produced in BHK-21 cells contained a poly(A) sequence averaging 50 to 55 nucleotides in length. Our results also indicated that the poly(A) is located at the 3' end of the viral RNA. Roller bottle cultures (5 x 108 cells) of BHK21 cells were infected with mengovirus (2), and the virus was purified as previously described (8). The viral RNA was labeled with [2,83H]adenosine (Schwarz/Mann, 30 Ci/mmol) by adding 500 ,Ci 2 h postinfection in 50 ml of culture fluid (2). For 32P labeling, 15 mCi of 32P was added to culture medium containing onetenth the normal phosphate concentration. Virus-infected culture fluid was harvested at 20 h after infection, and the virus was purified (8). Viral RNA was extracted with a sodium dodecyl sulfate (SDS), phenol and chloroform mixture (11), and the 35S RNA component was isolated by velocity sedimentation or sucrose gradient (2). -
743
Before the analysis for poly(A) sequences could be undertaken, it was first necessary to determine whether or not the purified 35S viral RNA was intact and free of small RNA fragments. The purified 35S viral RNA was analyzed by polyacrylamide gel electrophoresis in aqueous buffers containing SDS (Fig. 1A) and under denaturing conditions in 98% formamide (Fig. 1B) (15). The RNA was shown to be largely intact under either condition. Most importantly, small contaminating RNAs were not detected under denaturing conditions when RNA (-100,000 cpm) was electrophoresed for either 4 h (not shown) or 14 h. To isolate poly(A) sequences from mengovirus RNA, 32P-labeled 35S viral RNA was first digested with RNase T1 as previously described (15) and deproteinized (11), and the digested RNA was fractionated by affinity chromatography on 0.1-g oligodeoxythymidylic acid [oligo(dT)] cellulose columns (Collaborative Research, Inc.). The poly(A)-containing fragments were bound to the oligo(dT) cellulose in 0.5 M LiCl containing 0.01 M Tris-hydrochloride (pH 7.5) and 0.05% SDS (T-SDS buffer). The column was washed with 5 ml of the 0.5 M LiCl-T-SDS buffer and 5 ml of a 0.1 M LiClT-SDS buffer. Elution of the column with 10 ml of T-SDS buffer containing no LiCl yielded an RNA fraction (0.6% of total), which had a bJase composition of 94.1 + 0.6% AMP, 5.1 + 0.8% CMP, 0.1 + 0.1% GMP, and 0.7 + 0.1% UMP. The base composition was determined by the method of Wang and Duesberg (15) using RNase T2 hydrolysis, and the results were the average of three experiments. Since AMP and CMP migrate closely during electrophoresis at pH 3.5, it is conceivable that the high CMP content of the poly(A) could be due to a trailing
744
NOTES
J. VIROL.
K. Chatterjee, H. L. Bachrach, and J. Polatnick, Virology, in press) are located at the 3' end of the genomic RNA. If mengovirus poly(A) is I0 at the 3' end of the 35S viral RNA, the poly(A) tract generated by treatment of viral 35S RNA 8 with RNases A and T, should contain a 3' terminal adenosine residue. Complete hydro6lysis with alkali or RNase T2 would then pro04duce free adenosine as well as AMP. To test this, [3H]adenosine-labeled 35S viral RNA was BPB digested with RNases A and T1, and the poly(A) sequence was isolated by affinity chromatography on oligo(dT) cellulose. The poly(A) was digested with RNase T, and the digest was .- 3 analyzed by electrophoresis (15) to determine the amount of AMP and adenosine. The results of a typical experiment showed that the poly(A) 2 contained 18,281 cpm of L3H]AMP and 340 cpm of [3H]adenosine. Similar results were obtained by alkaline hydrolysis of the mengovirus poly(A) (81,341 cpm of [:3H]AMP and 1,489 cpm of [3H]adenosine). These results indicate that the poly(A) sequence is 3' terminal in mengo. virus RNA. 20 40 80 60 03 The size of the mengovirus poly(A) can be estimated from the ratio of AMP to adenosine. Distance Migrated (mm A value of 52 + 3 residues was obtained from an FIG. 1. Electrophoretic analysis of RNA. (A) [3H]adenosine-labeled mengo'virus RNA average of three experiments. This result agrees quite well with the size distribution of was purified and co-electrophoresed witih BHK 18S 0
and 28S 'IC-labeled rRNA (1) on 2.5% SDS-polyacrylamide gels according to Duesberg arid Vogt (5). Electrophoresis was for 6 h at 80 V, const'ant voltage. After electrophoresis, the gels were frozen in ethanoldry ice and sliced into 2-mm sections for roradioactivity determination (8). (B) [3H]Adenosine-laibeled mengovirus RNA was purified and co-elecitrophoresed with BHK 28S '4C-labeled rRNA (1) on 3'% polyacrylamide-98% formamide gels according too Duesberg and Vogt (5). The 28S rRNA was purifieci by velocity sedimentation on a linear sucrose gradie nt. Electrophoresis was for 14 h at 4 mA/gel, constc:nt current. The gel was processed for radioactivity determination as in (A).
of the AMP spot. Alternatively, the high CMP content could arise from a stretch o: f CMPs at the 5' end of the poly(A) sequence. The relative size of this poly(A) vwas determined by polyacrylamide-formamidEa gel electrophoresis (Fig. 2). The poly(A) fratction was found to be heterogeneous with RNA migrating slower and faster than tRNA. The nnajority of the poly(A) was smaller in size than tRNA. Digestion of 32P-labeled 35S m engovirus RNA with both RNases A and T, aind subsequent affinity chromatography on oliggo(dT) cel lulose (15) gave results similar to those described above. The poly(A) sequences in poliovirus RNA (14, 16) and foot-and-mouth disease virus RNA (N.
4,4
4s
o x
E 3
t /
> I - 2 3 o BPB
Dye
0D 20
60 100 0 Distance Migrated (mm)
FIG. 2. Polyacrylamide-formamide gel electrophoresis of RNase T,-resistant mengovirus RNA isolated by chromatography on oligo(dT) cellulose. The
RNAse T,-resistant mengovirus RNA fragments obtained by oligo(dT) cellulose chromatography were dissolved in water and a portion was electro-
phoresed on 12% polyacrylamide-98% formamide gels according to Duesberg and Vogt (5). Electrophoresis was for 3 h at 5 mA/gel, constant current.
['4C]uridine-labeled BHK tRNA was included as a marker indicated by an arrow. The gel was processed for radioactivity determination as in Fig. 1A.
VOL. 19, 1976
the poly(A) obtained by electrophoresis in 98% formamide-polyacrylamide gels (Fig. 2) in which most of the poly(A) was somewhat lower in molecular weight than tRNA (-80 nucleotides). Another way to estimate the average size of this poly(A) is to multiply the percentage of total mass of poly(A) by the estimated number of nucleotides in the viral RNA. Assuming the length of mengovirus RNA to be approximately 8,000 nucleotides based on a molecular weight estimate of 2.7 x 106 (4) and using the value of 0.6% as the percentage of total 35S viral RNA representing poly(A) sequences (obtained by affinity chromatography after RNase T, digestion, see above), an average poly(A) length was calculated to be about 50 nucleotides. This calculation also assumes that all viral RNA molecules contain a poly(A) tract, which has not yet been demonstrated. The results presented here suggest that mengovirus 35S RNA contains a poly(A) tract at its 3' terminus which is heterogeneous size, the average size estimated to be 50 to 55 residues. Our results support those of Spector and Baltimore (13), but not those obtained by Miller and Plagemann (9). Spector and Baltimore (13) estimated the size of mengovirus poly(A) as 50 to 70 nucleotides. Miller and Plagemann (9) estimated the size of mengovirus poly(A) to be 15 to 17 residues. In support of Miller and Plagemann (9), Porter et al. (12) were not able to detect large tracts of poly(A) by electrophoresis of RNase T, digests of encephalomyocarditis viral RNA in urea-polyacrylamide gels. Such a procedure should be able to detect poly(A) tracts containing 40 to 50 or more residues. It is difficult to reconcile the results obtained by Miller and Plagemann (9) and Porter et al. (12) on one hand with our results, and those of Spector and Baltimore (13) on the other. Possibly, the discrepancy is due to differences in methodology used to obtain the viral RNA and/ or techniques used to isolate the poly(A) tracts. The poly(A) sequence in poliovirus (14, 16) and foot-and-mouth disease virus (Chatterjee et al., Virology, in press) RNAs has been shown to be located at the 3' terminus of the genomic RNA. Our results obtained by complete hydrolysis by RNase T2 or alkali of the mengovirus poly(A) showed the presence of an adenosine. Given the specificity of RNases A and T1 used to produce the poly(A) and the mechanism of action of RNase T2 or alkali -during complete hy-
NOTES
745
drolysis of the poly(A), adenosine could only be produced from an RNA containing a 3' terminal poly(A) sequence. This work was supported in part by a grant (G-429) from The Robert A. Welch Foundation. Milton V. Marshall was a predoctoral fellow supported by The Rosalie B. Hite Foundation and Public Health Service Biology Training grant CA 5047 from the National Cancer Institute. LITERATURE CITED 1. Arlinghaus, R. B., W. Kaczmarczyk, and J. Polatnick. 1969. Electrophoretic characterization of foot-andmouth disease virus-specific ribonucleic acid. J. Virol. 4:712-718. 2. Arlinghaus, R. B., J. J. Syrewicz, and W. T. Loesch, Jr. 1973. RNA polymerase from mengovirus infected cells. Arch. Gesamte Virusforsch. 38:17-28. 3. Armstrong, J. A., M. Edmonds, H. Nakazato, B. S. Phillips, and M. H. Vaughan. 1972. Polyadenylic acid sequences in the virion RNA of poliovirus and eastern equine encephalitis virus. Science 176:526-528. 4. Burness, A. T. H. 1970. Ribonucleic acid content of encephalomyocarditis virus. J. Gen. Virol. 6:373-380. 5. Duesberg, P., and P. K. Vogt. 1973. Gel electrophoresis of avian leukosis and sarcoma viral RNA in formamide: comparison with other viral and cellular RNA species. J. Virol. 12:594-599. 6. Gillespie, D., K. Takemoto, M. Robert, and R. C. Gallo. 1973. Polyadenylic acid in visna virus RNA. Science 179:1328-1330. 7. Johnston, R., and H. R. Bose. 1972. Correlation of messenger RNA function with adenylate rich sequences in genome of single stranded RNA viruses. Proc. Natl. Acad. Sci. U.S.A. 69:1514-1516. 8. Loesch, W. T., and R. B. Arlinghaus. 1974. Polypeptides associated with the 250S mengovirus-induced RNA polymerase structure. Arch. Gesamte Virusforsch. 46:253-268. 9. Miller, R. L., and P. G. W. Plagemann. 1972. Purification of mengovirus and identification of an A-rich segment. J. Gen. Virol. 17:349-353. 10. Nair, C. N., and M. J. Owens. 1974. Preliminary observations pertaining to polyadenylation of rhinovirus RNA. J. Virol. 13:535-537. 11. Perry, R. P., J. LaTorre, D. E. Kelley, and J. R. Greenberg. 1972. On the lability of poly(A) sequences during extraction of messenger RNA from polyribosomes. Biochim. Biophys. Acta 262:220-226. 12. Porter, A., N. Carey, and P. Fellner. 1974. Presence of a large poly(rC) tract within the RNA of encephalomyocarditis virus. Nature (London) 248:675-678. 13. Spector, D., and D. Baltimore. 1975. Poly(A) of mengovirus RNA. J. Virol. 16:1081-1084. 14. Spector, D. H., and D. Baltimore. 1974. Requirement of 3'-terminal polyadenylic acid for the infectivity of poliovirus RNA. Proc. Natl. Acad. Sci. U.S.A. 71:2983-2987. 15. Wang, L., and P. Duesberg. 1974. Properties and location of poly(A) in Rous sarcoma virus RNA. J. Virol. 14:1515-1529. 16. Yogo, Y., and E. Wimmer. 1972. Polyadenylic acid at the 3'-terminus of poliovirus RNA. Proc. Natl. Acad. Sci. U.S.A. 69:1877-1882.
Vol. 19, No. 2 Printed in U.S.A.
NOTES Polyadenylic Acid in the Genomic RNA of Mengovirus MILTON V. MARSHALL AND RALPH B. ARLINGHAUS* Departments of Biology* and Biochemistry, The University of Texas System Cancer Center, M. D. Anderson Hospital and Tumor Institute, Houston, Texas 77030 Received for publication 27 February 1976
The polyadenylic acid contained in 35S mengovirus RNA produced in infected BHK-21 cells contained approximately 94% AMP and was estimated to contain an average of 50 to 55 nucleotides. The polyadenylic acid is placed at the 3'-end of the genomic RNA based on the presence of significant levels of [3H]adenosine in complete alkali or RNase T2 digests of polyadenylic acid from [3H]adenosinelabeled 35S viral RNA.
Polyadenylic acid [poly(A)] sequences have been found in picornavirus RNA genomes such as poliovirus (3, 14, 16), encephalomyocarditis virus (6), Columbia S-K virus (7), mengovirus (9, 13), foot-and-mouth disease virus (N. K. Chatteijee, H. L. Bachrach, and J. Polatnick, Virology, in press), and rhinovirus (10). These poly(A) sequences range in size from 50 to 100 nucleotides in length. Some controversy exists with respect to the size of the poly(A) sequence in mengovirus RNA. Miller and Plagemann (9) have reported that mengovirus RNA produced in Novikoff rat hepatoma cells contained a short tract of poly(A) (15 to 17 residues). In contrast to this, Spector and Baltimore (13) showed that mengovirus RNA produced in L cells and HeLa cells contained a longer poly(A) sequence (50 to 70 residues). In the work described here, mengovirus RNA produced in BHK-21 cells contained a poly(A) sequence averaging 50 to 55 nucleotides in length. Our results also indicated that the poly(A) is located at the 3' end of the viral RNA. Roller bottle cultures (5 x 108 cells) of BHK21 cells were infected with mengovirus (2), and the virus was purified as previously described (8). The viral RNA was labeled with [2,83H]adenosine (Schwarz/Mann, 30 Ci/mmol) by adding 500 ,Ci 2 h postinfection in 50 ml of culture fluid (2). For 32P labeling, 15 mCi of 32P was added to culture medium containing onetenth the normal phosphate concentration. Virus-infected culture fluid was harvested at 20 h after infection, and the virus was purified (8). Viral RNA was extracted with a sodium dodecyl sulfate (SDS), phenol and chloroform mixture (11), and the 35S RNA component was isolated by velocity sedimentation or sucrose gradient (2). -
743
Before the analysis for poly(A) sequences could be undertaken, it was first necessary to determine whether or not the purified 35S viral RNA was intact and free of small RNA fragments. The purified 35S viral RNA was analyzed by polyacrylamide gel electrophoresis in aqueous buffers containing SDS (Fig. 1A) and under denaturing conditions in 98% formamide (Fig. 1B) (15). The RNA was shown to be largely intact under either condition. Most importantly, small contaminating RNAs were not detected under denaturing conditions when RNA (-100,000 cpm) was electrophoresed for either 4 h (not shown) or 14 h. To isolate poly(A) sequences from mengovirus RNA, 32P-labeled 35S viral RNA was first digested with RNase T1 as previously described (15) and deproteinized (11), and the digested RNA was fractionated by affinity chromatography on 0.1-g oligodeoxythymidylic acid [oligo(dT)] cellulose columns (Collaborative Research, Inc.). The poly(A)-containing fragments were bound to the oligo(dT) cellulose in 0.5 M LiCl containing 0.01 M Tris-hydrochloride (pH 7.5) and 0.05% SDS (T-SDS buffer). The column was washed with 5 ml of the 0.5 M LiCl-T-SDS buffer and 5 ml of a 0.1 M LiClT-SDS buffer. Elution of the column with 10 ml of T-SDS buffer containing no LiCl yielded an RNA fraction (0.6% of total), which had a bJase composition of 94.1 + 0.6% AMP, 5.1 + 0.8% CMP, 0.1 + 0.1% GMP, and 0.7 + 0.1% UMP. The base composition was determined by the method of Wang and Duesberg (15) using RNase T2 hydrolysis, and the results were the average of three experiments. Since AMP and CMP migrate closely during electrophoresis at pH 3.5, it is conceivable that the high CMP content of the poly(A) could be due to a trailing
744
NOTES
J. VIROL.
K. Chatterjee, H. L. Bachrach, and J. Polatnick, Virology, in press) are located at the 3' end of the genomic RNA. If mengovirus poly(A) is I0 at the 3' end of the 35S viral RNA, the poly(A) tract generated by treatment of viral 35S RNA 8 with RNases A and T, should contain a 3' terminal adenosine residue. Complete hydro6lysis with alkali or RNase T2 would then pro04duce free adenosine as well as AMP. To test this, [3H]adenosine-labeled 35S viral RNA was BPB digested with RNases A and T1, and the poly(A) sequence was isolated by affinity chromatography on oligo(dT) cellulose. The poly(A) was digested with RNase T, and the digest was .- 3 analyzed by electrophoresis (15) to determine the amount of AMP and adenosine. The results of a typical experiment showed that the poly(A) 2 contained 18,281 cpm of L3H]AMP and 340 cpm of [3H]adenosine. Similar results were obtained by alkaline hydrolysis of the mengovirus poly(A) (81,341 cpm of [:3H]AMP and 1,489 cpm of [3H]adenosine). These results indicate that the poly(A) sequence is 3' terminal in mengo. virus RNA. 20 40 80 60 03 The size of the mengovirus poly(A) can be estimated from the ratio of AMP to adenosine. Distance Migrated (mm A value of 52 + 3 residues was obtained from an FIG. 1. Electrophoretic analysis of RNA. (A) [3H]adenosine-labeled mengo'virus RNA average of three experiments. This result agrees quite well with the size distribution of was purified and co-electrophoresed witih BHK 18S 0
and 28S 'IC-labeled rRNA (1) on 2.5% SDS-polyacrylamide gels according to Duesberg arid Vogt (5). Electrophoresis was for 6 h at 80 V, const'ant voltage. After electrophoresis, the gels were frozen in ethanoldry ice and sliced into 2-mm sections for roradioactivity determination (8). (B) [3H]Adenosine-laibeled mengovirus RNA was purified and co-elecitrophoresed with BHK 28S '4C-labeled rRNA (1) on 3'% polyacrylamide-98% formamide gels according too Duesberg and Vogt (5). The 28S rRNA was purifieci by velocity sedimentation on a linear sucrose gradie nt. Electrophoresis was for 14 h at 4 mA/gel, constc:nt current. The gel was processed for radioactivity determination as in (A).
of the AMP spot. Alternatively, the high CMP content could arise from a stretch o: f CMPs at the 5' end of the poly(A) sequence. The relative size of this poly(A) vwas determined by polyacrylamide-formamidEa gel electrophoresis (Fig. 2). The poly(A) fratction was found to be heterogeneous with RNA migrating slower and faster than tRNA. The nnajority of the poly(A) was smaller in size than tRNA. Digestion of 32P-labeled 35S m engovirus RNA with both RNases A and T, aind subsequent affinity chromatography on oliggo(dT) cel lulose (15) gave results similar to those described above. The poly(A) sequences in poliovirus RNA (14, 16) and foot-and-mouth disease virus RNA (N.
4,4
4s
o x
E 3
t /
> I - 2 3 o BPB
Dye
0D 20
60 100 0 Distance Migrated (mm)
FIG. 2. Polyacrylamide-formamide gel electrophoresis of RNase T,-resistant mengovirus RNA isolated by chromatography on oligo(dT) cellulose. The
RNAse T,-resistant mengovirus RNA fragments obtained by oligo(dT) cellulose chromatography were dissolved in water and a portion was electro-
phoresed on 12% polyacrylamide-98% formamide gels according to Duesberg and Vogt (5). Electrophoresis was for 3 h at 5 mA/gel, constant current.
['4C]uridine-labeled BHK tRNA was included as a marker indicated by an arrow. The gel was processed for radioactivity determination as in Fig. 1A.
VOL. 19, 1976
the poly(A) obtained by electrophoresis in 98% formamide-polyacrylamide gels (Fig. 2) in which most of the poly(A) was somewhat lower in molecular weight than tRNA (-80 nucleotides). Another way to estimate the average size of this poly(A) is to multiply the percentage of total mass of poly(A) by the estimated number of nucleotides in the viral RNA. Assuming the length of mengovirus RNA to be approximately 8,000 nucleotides based on a molecular weight estimate of 2.7 x 106 (4) and using the value of 0.6% as the percentage of total 35S viral RNA representing poly(A) sequences (obtained by affinity chromatography after RNase T, digestion, see above), an average poly(A) length was calculated to be about 50 nucleotides. This calculation also assumes that all viral RNA molecules contain a poly(A) tract, which has not yet been demonstrated. The results presented here suggest that mengovirus 35S RNA contains a poly(A) tract at its 3' terminus which is heterogeneous size, the average size estimated to be 50 to 55 residues. Our results support those of Spector and Baltimore (13), but not those obtained by Miller and Plagemann (9). Spector and Baltimore (13) estimated the size of mengovirus poly(A) as 50 to 70 nucleotides. Miller and Plagemann (9) estimated the size of mengovirus poly(A) to be 15 to 17 residues. In support of Miller and Plagemann (9), Porter et al. (12) were not able to detect large tracts of poly(A) by electrophoresis of RNase T, digests of encephalomyocarditis viral RNA in urea-polyacrylamide gels. Such a procedure should be able to detect poly(A) tracts containing 40 to 50 or more residues. It is difficult to reconcile the results obtained by Miller and Plagemann (9) and Porter et al. (12) on one hand with our results, and those of Spector and Baltimore (13) on the other. Possibly, the discrepancy is due to differences in methodology used to obtain the viral RNA and/ or techniques used to isolate the poly(A) tracts. The poly(A) sequence in poliovirus (14, 16) and foot-and-mouth disease virus (Chatterjee et al., Virology, in press) RNAs has been shown to be located at the 3' terminus of the genomic RNA. Our results obtained by complete hydrolysis by RNase T2 or alkali of the mengovirus poly(A) showed the presence of an adenosine. Given the specificity of RNases A and T1 used to produce the poly(A) and the mechanism of action of RNase T2 or alkali -during complete hy-
NOTES
745
drolysis of the poly(A), adenosine could only be produced from an RNA containing a 3' terminal poly(A) sequence. This work was supported in part by a grant (G-429) from The Robert A. Welch Foundation. Milton V. Marshall was a predoctoral fellow supported by The Rosalie B. Hite Foundation and Public Health Service Biology Training grant CA 5047 from the National Cancer Institute. LITERATURE CITED 1. Arlinghaus, R. B., W. Kaczmarczyk, and J. Polatnick. 1969. Electrophoretic characterization of foot-andmouth disease virus-specific ribonucleic acid. J. Virol. 4:712-718. 2. Arlinghaus, R. B., J. J. Syrewicz, and W. T. Loesch, Jr. 1973. RNA polymerase from mengovirus infected cells. Arch. Gesamte Virusforsch. 38:17-28. 3. Armstrong, J. A., M. Edmonds, H. Nakazato, B. S. Phillips, and M. H. Vaughan. 1972. Polyadenylic acid sequences in the virion RNA of poliovirus and eastern equine encephalitis virus. Science 176:526-528. 4. Burness, A. T. H. 1970. Ribonucleic acid content of encephalomyocarditis virus. J. Gen. Virol. 6:373-380. 5. Duesberg, P., and P. K. Vogt. 1973. Gel electrophoresis of avian leukosis and sarcoma viral RNA in formamide: comparison with other viral and cellular RNA species. J. Virol. 12:594-599. 6. Gillespie, D., K. Takemoto, M. Robert, and R. C. Gallo. 1973. Polyadenylic acid in visna virus RNA. Science 179:1328-1330. 7. Johnston, R., and H. R. Bose. 1972. Correlation of messenger RNA function with adenylate rich sequences in genome of single stranded RNA viruses. Proc. Natl. Acad. Sci. U.S.A. 69:1514-1516. 8. Loesch, W. T., and R. B. Arlinghaus. 1974. Polypeptides associated with the 250S mengovirus-induced RNA polymerase structure. Arch. Gesamte Virusforsch. 46:253-268. 9. Miller, R. L., and P. G. W. Plagemann. 1972. Purification of mengovirus and identification of an A-rich segment. J. Gen. Virol. 17:349-353. 10. Nair, C. N., and M. J. Owens. 1974. Preliminary observations pertaining to polyadenylation of rhinovirus RNA. J. Virol. 13:535-537. 11. Perry, R. P., J. LaTorre, D. E. Kelley, and J. R. Greenberg. 1972. On the lability of poly(A) sequences during extraction of messenger RNA from polyribosomes. Biochim. Biophys. Acta 262:220-226. 12. Porter, A., N. Carey, and P. Fellner. 1974. Presence of a large poly(rC) tract within the RNA of encephalomyocarditis virus. Nature (London) 248:675-678. 13. Spector, D., and D. Baltimore. 1975. Poly(A) of mengovirus RNA. J. Virol. 16:1081-1084. 14. Spector, D. H., and D. Baltimore. 1974. Requirement of 3'-terminal polyadenylic acid for the infectivity of poliovirus RNA. Proc. Natl. Acad. Sci. U.S.A. 71:2983-2987. 15. Wang, L., and P. Duesberg. 1974. Properties and location of poly(A) in Rous sarcoma virus RNA. J. Virol. 14:1515-1529. 16. Yogo, Y., and E. Wimmer. 1972. Polyadenylic acid at the 3'-terminus of poliovirus RNA. Proc. Natl. Acad. Sci. U.S.A. 69:1877-1882.
