Sindbis virus, a togavirus, contains an infectious, single stranded RNA with a molecular weight of 4.3 x 106 (498), which in infected cells serves as a messenger RNA for the translation of the virus onstructural proteins. Also present in infected cells is anRNA with a molecular weight of 1.6 x 106 (268) which consists of the 3' terminal third of the 49S RNA and functions as a messenger RNAfor the translation of the virus structural proteins. In this thesis,three features of these RNA species of Sindbis virus are studied:the 3' terminal poly A tracts on the RNAs and the corresponding polyU tracts on the complementary strand of RNA; the methylation ofcytidine residues in the RNA; and the circularization of the 49S RNAmolecule.
Both 49S and 26S RNA contain 3' terminal stretches of polyadenylicacid (poly A), which are heterogeneous in size, having a mean lengthof 70 nucleotides and a size range of from 40 to 200 nucleotides.Poly A isolated from Sindbis virion 49S RNA grown in chicken, hamsterand mosquito culture cells have similar size distributions. Althoughmost 49S and 26S RNA molecules contain poly A, a small fraction ofintact 49S and 26S RNA molecules contain little poly A. The fact thatthe fraction of 49S which lacks poly A is only 10% to 20% as infectiousas the fraction which contains poly A suggests that poly A is essentialfor replication of the virus. Sindbis virus double stranded RNAspecies also contain poly A with a size distribution similar to thatof poly A from viral single stranded RNA. The double stranded RNA species also contain stretches of polyuridylic acid (poly U) whichare on the minus strand and have a size distribution identical toSindbis virus poly A. This indicates that the poly A in Sindbisvirus RNA is synthesized by transcription of a poly U template by thevirus transcriptase.
Sindbis virus intracellular 26S and 49S RNA contain internal5-methyl cytidine (m5c) residues. Sindbis virion 49S RNA containsmuch less m5c than intracellular 49S RNA. In the 26S RNA, m5c residuesoccur in five oligonucleotides, which are found distributed betweentwo locations, one approximately 4000 nucleotides from the 3' end andthe other about 1200 nucleotides from the 31 end (out of a total lengthof 5000 nucleotides). The distribution of label between these twolocations suggests that each contains at least two of the methylatedsequences. It thus appears that there are five specific sites formethylation on the 26S RNA. However, only a minority of these sitesare modified. Polysomal and nonpolysomal 26S RNA contain equal amountsof m5c while polysomal 49S RNA contains 60% to 80% more m5c thannonpolysomal 49S RNA, indicating that m5c may have a function intranslation.
Sindbis virus 49S RNA is capable of assuming a circular configurationthrough the hydrogen bonding of complementary nucleotide sequenceslocated at the 5' and 3' ends of the 49s RNA molecule. The circularand linear forms of 49S RNA are separable on sucrose gradientscontaining 0.01 M NaCl. Sindbis virus 49S RNA extracted from virionsis completely in the circular form. The melting temperature (Tm) of the circles is 39.5°C in 0.023 M NaCl and 53.5°C in 0.1 M NaCl. The 6Hfor circularization is -160 kcal/mole and the ΔS for circularization isapproximately 500 eu. These parameters indicate that the length of thedouble-stranded region which is formed upon circularization of themolecule is most likely short, on the order of 10 to 20 nucleotides.Our data indicate that extensive mismatching in this double strandedregion is unlikely. Intact linear 49S RNA molecules readily renatureto form circles under appropriate conditions, the energy of activationfor this process being 42.6 kcal/mole. From the measured rate constantsfor circularization, it is clear that Sindbis virus 49S RNA willcircularize readily under physiological conditions of temperature andionic strength. The virion RNA from Semliki Forest virus also formscircles whose Tm is very similar to that of Sindbis virus RNA circles, suggesting that the sequences involved in circularization have beenconserved.
