Chromatography of arbovirus ribonucleic acid forms on columns of benzoylated-diethylaminoethyl cellulose.
Benzoylated-diethylaminoethyl cellulose (BD-cellulose) column chromatography was found to be useful in resolving most of the ribonucleic acid (RNA) forms from the replicative cycle of group A arbovirus Semliki Forect virus (SFV). The elution patterns were independent of molecular weight and appeared to be related to the degree of secondary structure in the molecule. Fractions of RNA were taken from a sucrose density gradient of cytoplasmic extracts of SFV-infected chick cells pretreated with actinomycin D. In a linear salt gradient, 16S material cochromatographed with the rapidly eluted ribonuclease resistant core of the double-stranded SFV-RNA and with the homopolymer duplex polyinosinic acid: polycytidylic acid. This fraction, therefore, probably contains an SFV-RNA form similar to the completely double stranded replicative form (RF) of several RNA viruses and bacteriophages. Faster moving (>20S) sucrose gradient fractions eluted more slowly, suggesting a decreasing proportion of secondary structure with increasing sedimentation value. The fractions, therefore, seemed to contain replicative intermediate (RI) structures. The two single stranded forms of SFV-RNA (42S and 26S) could only be eluted from BD-cellulose in the presence of urea or dimethyl sulfoxide, suggesting the presence of minimal secondary structure. Under these conditions, the single-stranded viral RNA forms could not be resolved. Molecular sieve chromatography of the single-stranded RNA forms, performed by passage through an agarose column, also failed to resolve these forms. The viral RNA forms containing a high degree of secondary structure, probably the RF and the RI, could, therefore, be rapidly separated from each other and from the single-stranded forms.
D000818:Animals; D001103:Arboviruses / Q000032:analysis*; D002499:Centrifugation, Density Gradient; D002642:Chick Embryo; D002848:Chromatography, DEAE-Cellulose; D005347:Fibroblasts; D066298:In Vitro Techniques; D008722:Methods; D010759:Phosphorus Isotopes; D012367:RNA, Viral / Q000032:analysis* / Q000302:isolation & purification; D013395:Sucrose; D014316:Tritium; D014529:Uridine; D014779:Virus Replication
D010759:Phosphorus Isotopes; D012367:RNA, Viral; D014316:Tritium; D013395:Sucrose; D014529:Uridine
J Mol Biol. 1965 Jul;12(3):829-42
4955314
Proc Natl Acad Sci U S A. 1965 Sep;54(3):982-7
5217476
Proc Natl Acad Sci U S A. 1966 Aug;56(2):440-6
5229967
Nature. 1967 Jan 28;213(5074):365-7
6067598
J Mol Biol. 1967 Jun 28;26(3):537-40
5339798
Biochemistry. 1967 Oct;6(10):3043-56
6056973
Biochemistry. 1968 Jul;7(7):2629-34
5660079
J Virol. 1968 Jun;2(6):547-52
5748743
J Virol. 1968 Jun;2(6):558-66
5697470
J Virol. 1967 Apr;1(2):374-83
5630381
J Mol Biol. 1963 Jul;7:43-54
13978804
Science. 1964 Oct 23;146(3643):527-30
14190242
Biochemistry. 1964 Aug;3:1015-21
14220659