Student Theses and Dissertations
Date of Award
1969
Document Type
Thesis
Degree Name
Doctor of Philosophy (PhD)
Thesis Advisor
Igor Tamm
Keywords
Semliki Forest virus, nucleocapsid structure, viral budding, electron microscopy, RNA content, virus assembly
Abstract
Semliki Forest virus is a group A arthropod-borne virus which contains RNA and possesses a lipoprotein envelope. It can be transmitted among animals by mosquitoes in which it multiplies, but its natural host is not known. The virus grows to high titer in a number of types of vertebrate cells in culture. The growth of Semliki Forest virus was studied with the electron microscope during a single cycle of viral replication in chick embryo cells. The spherical virus particle consists of a nucleoid, or nucleocapsid, 280 A in diameter, closely wrapped in an envelope which consists of a unit membrane 75 A thick coated on its outer surface with projections 110 A long. There appears to be a narrow 10-30 A space between the nucleocapsid and the envelope. The diameter of the virus particle, excluding the projections, is approximately 500 A, and thus the whole virus particle is about 700 A in diameter. After a latent period of 2 to 3 hours, infectious virus is produced at a nearly constant rate of approximately 200 plaque-forming units/cell per hour, until the 9th hour. During the period of rapid production of virus, individual free virus nucleocapsids are found scattered in the cytoplasmic matrix. The nucleocapsids appear to migrate singly to the plasma membrane, or to the membranes of certain vacuoles, where complete virus particles are formed by a budding process. An envelope, consisting of a portion of the membrane covered with projections, encloses the nucleocapsid as it is extruded into the extracellular space or the interior of a vacuole. Most developing virus particles are seen at the plasma membrane; fewer are found at the membranes of vacuoles. Small vesicles, consisting of buds of membrane, are found projecting into large cytoplasmic vacuoles or into the extracellular space at the plasma membrane. These vesicles do not contain nucleocapsids, and there is little evidence of the projections which virus envelopes have. The exact nature of the vesicles is unknown, but they may represent aberrant envelopes. Late in the growth cycle, nucleocapsids are increasingly found lining the cytoplasmic surface of round or tubular vacuoles. These vacuoles often contain long cylindrical structures which-appear to be formed from a membrane covered with amorphous material. Because the vacuoles lined with nucleocapsids appear in large numbers only at late stages in the viral growth cycle, they do not appear to have a functional role in the production of Semliki Forest virus particles. Nucleocapsids of Semliki Forest virus have been isolated and purified from homogenates of infected chick embryo cells. The purification procedure consists of a preliminary centrifugation in a sucrose gradient, followed by dialysis against a buffer lacking magnesium. Ribosomes contaminating the nucleocapsids are dissociated into subunits by this means. The dialyzed material is resedimented twice in succession in sucrose gradients, to separate the nucleocapsids from the ribosomal subunits and to concentrate the purified nucleocapsids. Purified, negatively-stained nucleocapsids are roughly spherical and 400 A in diameter, slightly larger than nucleocapsids in thin sections (280 A). The reason for this discrepancy is not known. Nucleocapsids are penetrated by the negative stain (phosphotungstate) in some preparations but not in others. A mottled surface structure indicates that the nucleocapsids may have a subunit structure, but there is no clear indication of symmetry. Unfixed nucleocapsids disintegrate in high concentrations of cesium chloride, but after formaldehyde fixation they can be banded to an equilibrium density of 147 gm/cc in cesium chloride gradients. In potassium tartrate gradients nucleocapsids band at an equilibrium density of 1.34 gm/cc, but the RNA within nucleocapsids is fragmented by this procedure. The high buoyant density and the ultraviolet absorbance profile of nucleocapsids indicate an RNA content greater than 30% of the total mass. Nucleocapsids contain 45 S RNA, found also in complete virus. Two protein bands are resolved on gel electropherograms of purified nucleocapsids. The major protein has a molecular weight of approximately 35,000, and the minor protein, 70,000. The latter protein is present only in small amounts, and may be a dimer of the major protein. Purified virus contains these two proteins, in the same proportion as in the nucleocapsids, and, in addition, a major protein of molecular weight approximately 55,000. This protein is probably located in the virus envelope. The RNA within nucleocapsids is accessible to digestion by pancreatic ribonuclease. The nucleocapsids differ in this feature from most small RNA-containing viruses, which are resistant to ribonuclease. The nucleocapsid structure disintegrates upon digestion of the RNA by ribonuclease.
License and Reuse Information
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Recommended Citation
Acheson, Nicholas Hill, "The Structure and Replication of Semliki Forest Virus, A Mosquito-Borne Animal Virus" (1969). Student Theses and Dissertations. 552.
https://digitalcommons.rockefeller.edu/student_theses_and_dissertations/552
Comments
A thesis presented to the faculty of The Rockefeller University in partial fulfillment of the requirements for the degree of Doctor of Philosophy