Biochemical and Biophysical Studies on the RNA Species of Sindbis Virus

Citation

Frey, Teryl Kenneth (1978) Biochemical and Biophysical Studies on the RNA Species of Sindbis Virus. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/18xf-7p07. https://resolver.caltech.edu/CaltechTHESIS:03032017-150817762

Abstract

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 an RNA 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 RNA for 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 poly U tracts on the complementary strand of RNA; the methylation of cytidine residues in the RNA; and the circularization of the 49S RNA molecule.

Both 49S and 26S RNA contain 3' terminal stretches of polyadenylic acid (poly A), which are heterogeneous in size, having a mean length of 70 nucleotides and a size range of from 40 to 200 nucleotides. Poly A isolated from Sindbis virion 49S RNA grown in chicken, hamster and mosquito culture cells have similar size distributions. Although most 49S and 26S RNA molecules contain poly A, a small fraction of intact 49S and 26S RNA molecules contain little poly A. The fact that the fraction of 49S which lacks poly A is only 10% to 20% as infectious as the fraction which contains poly A suggests that poly A is essential for replication of the virus. Sindbis virus double stranded RNA species also contain poly A with a size distribution similar to that of poly A from viral single stranded RNA. The double stranded RNA species also contain stretches of polyuridylic acid (poly U) which are on the minus strand and have a size distribution identical to Sindbis virus poly A. This indicates that the poly A in Sindbis virus RNA is synthesized by transcription of a poly U template by the virus transcriptase.

Sindbis virus intracellular 26S and 49S RNA contain internal 5-methyl cytidine (m⁵c) residues. Sindbis virion 49S RNA contains much less m⁵c than intracellular 49S RNA. In the 26S RNA, m⁵c residues occur in five oligonucleotides, which are found distributed between two locations, one approximately 4000 nucleotides from the 3' end and the other about 1200 nucleotides from the 31 end (out of a total length of 5000 nucleotides). The distribution of label between these two locations suggests that each contains at least two of the methylated sequences. It thus appears that there are five specific sites for methylation on the 26S RNA. However, only a minority of these sites are modified. Polysomal and nonpolysomal 26S RNA contain equal amounts of m⁵c while polysomal 49S RNA contains 60% to 80% more m⁵c than nonpolysomal 49S RNA, indicating that m⁵c may have a function in translation.

Sindbis virus 49S RNA is capable of assuming a circular configuration through the hydrogen bonding of complementary nucleotide sequences located at the 5' and 3' ends of the 49s RNA molecule. The circular and linear forms of 49S RNA are separable on sucrose gradients containing 0.01 M NaCl. Sindbis virus 49S RNA extracted from virions is completely in the circular form. The melting temperature (T_m) of the circles is 39.5°C in 0.023 M NaCl and 53.5°C in 0.1 M NaCl. The 6H for circularization is -160 kcal/mole and the ΔS for circularization is approximately 500 eu. These parameters indicate that the length of the double-stranded region which is formed upon circularization of the molecule is most likely short, on the order of 10 to 20 nucleotides. Our data indicate that extensive mismatching in this double stranded region is unlikely. Intact linear 49S RNA molecules readily renature to form circles under appropriate conditions, the energy of activation for this process being 42.6 kcal/mole. From the measured rate constants for circularization, it is clear that Sindbis virus 49S RNA will circularize readily under physiological conditions of temperature and ionic strength. The virion RNA from Semliki Forest virus also forms circles whose T_m is very similar to that of Sindbis virus RNA circles, suggesting that the sequences involved in circularization have been conserved.

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