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Replication of Sindbis Virus II. Multiple Forms of Double-stranded RNA Isolated from Infected Cells

Summons, Daniel T. and Strauss, James H. (1972) Replication of Sindbis Virus II. Multiple Forms of Double-stranded RNA Isolated from Infected Cells. Journal of Molecular Biology, 71 (3). pp. 615-631. ISSN 0022-2836. http://resolver.caltech.edu/CaltechAUTHORS:20160620-145903296

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Abstract

Three species of double-stranded RNA (replicative forms) have been isolated from cells infected with Sindbis virus after treating the extracts with pancreatic ribonuclease. Their molecular weights have been estimated to be 8.8 x 10^6, 6.6 x 10^6 and 2.9 x 10^6 daltons (RFI, RFII and RFIII, respectively). RFII and RFIII were found to exist in a one-to-one molar ratio throughout the infection cycle, although the total amounts of these replicative forms varied. The molar ratio of RF1 to the other two replicative forms was different at various times after infection. However, in the middle of the virus life-cycle, from two to six hours post-infection, this ratio was constant at 0.4 to 1. Sedimentation analysis of pulse-labeled RNA from Sindbis-infected cells indicated that all replicative intermediates had molecular weights greater than or equal to 8.8 x 10^6 daltons. Since all three replicative forms were liberated from the peak of replicative intermediates by treatment with ribonuclease, this suggested that RFII and RFIII were joined together before digestion. The constant one-to-one molar ratio of RFII and RFIII supported this hypothesis. Kinetic experiments demonstrated that while RFI and RFIII were labeled equally rapidly, RFII was labeled some 20 to 50 times more slowly. Hybridization competition experiments showed that the minus strand of RFIII is complementary to 26 s RNA, a species of single-stranded RNA found in infected cells, and that the minus strand of RFII is complementary to the remainder of the 49 s viral genome. We postulate, therefore, that a precursor-product relation exists between RFI and 49 s RNA, between RFIII and 26 s RNA and between RFII and a third species of Sindbis-specific RNA, which we have not yet detected. Our results are consistent with the hypothesis that two classes of replicative intermediates exist in Sindbis-infected cells. One serves as the template for the synthesis of 49 s RNA, and the second manufactures two smaller segments of viral-specific RNA.


Item Type:Article
Related URLs:
URLURL TypeDescription
http://dx.doi.org/10.1016/S0022-2836(72)80027-5DOIArticle
http://www.sciencedirect.com/science/article/pii/S0022283672800275PublisherArticle
Additional Information:© 1972 Elsevier Ltd. Received 14 February 1972, and in revised form 10 July 1972. We are pleased to acknowledge the expert technical assistance of Edith M. Lenches and Mildred Quisenberry Hoover. We thank Drs Ellen G. Strauss and John S. Pierce for their helpful editorial advice for the construction of this manuscript. This investigation was supported in part by the National Science Foundation grant no. GB13608 and by the U.S. Public Health Service grant no. GM06965. One of us (D. T. S.) was supported by the National Institutes of Health training grant no. 5-Tl-GM-86.
Funders:
Funding AgencyGrant Number
NSFGB-13608
NIHGM-06965
NIH Predoctoral Fellowship5-Tl-GM-86
Record Number:CaltechAUTHORS:20160620-145903296
Persistent URL:http://resolver.caltech.edu/CaltechAUTHORS:20160620-145903296
Official Citation:Daniel T. Summons, James H. Steaitss, Replication of Sindbis virus, Journal of Molecular Biology, Volume 71, Issue 3, 1972, Pages 615-631, ISSN 0022-2836, http://dx.doi.org/10.1016/S0022-2836(72)80027-5. (http://www.sciencedirect.com/science/article/pii/S0022283672800275)
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:68543
Collection:CaltechAUTHORS
Deposited By: Donna Wrublewski
Deposited On:22 Jun 2016 04:44
Last Modified:22 Jun 2016 04:44

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