Student Theses and Dissertations

Date of Award

1968

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

Abstract

The simian parainfluenza virus SV5 is a member of myxovirus subgroup II, which includes parainfluenza, mumps, and Newcastle disease viruses. These viruses are 120-500 mi in diameter, and consist of an envelope covered with surface projections and an internal ribonucleoprotein component which is a single-stranded helix, 150-180 A in diameter, A high yield of infective SV5 is produced by primary cultures of rhesus monkey kidney (MK) cells, and the virus-cell interaction is moderate: infected cells divide normally, exhibit little cyctopathic effect, and cellular macromolecular synthesis is not inhibited.

An electron microscopic study of SV5 replication in MK cells was undertaken. Virus adsorbs to the cell surface and is then taken into cells by phagocytosis. Virus-induced morphological changes appear only in the cytoplasm of infected cells. The helical nucleocapsid of the virus appears to form in the cytoplasmic matrix and align under regions of the cell membrane which acquire viral surface projections. Assembly and release of virus particles at the cell surface occur by a budding process involving incorporation into the viral envelope of a unit membrane, which is continuous with and morphologically identical to that of the host cell. Both spherical and filamentous virus particles are formed. Filaments frequently contain nucleocapsid in a regular spiral which extends throughout their length. SV5 causes minimal cytopathic changes in MK cells, and there appears to be a balance between the rate of synthesis of nucleocapsid and its continuous release within mature virus particles.

Under certain conditions, a gradual accumulation of nucleocapsid is seen in the cytoplasm of infected cells. This observation suggested an approach to the isolation of SV5 nucleocapsid. Equilibrium sedimentation in cesium chloride gradients was used to purify nucleocapsid released from cells by osmotic shock. The length distribution of the released nucleocapsid shows a sharp peak with a mean of 1.02 |i, and it is probable that this length contains one SV5 genome. Chemical determinations indicate that the nucleocapsid is a ribonucleoprotein with an RNA content of 4„l%o The length distribution of the nucleocapsid of Newcastle disease virus closely resembles that obtained for SV5. On the basis of these and other results, it seems likely that /-^ 1 |j, is the unit length of the nucleocapsid of all subgroup II myxoviruses.

SV5 RNA was isolated from purified nucleocapsid or from virus purified equilibrium zonal centrifugation in a potassium tartrate gradient, in which the virus bands at a density of 1.22-1.23. The RNA was dissociated frora protein by treatment with sodium dodecyl sulfate, and purified by sedimentation in a sucrose density gradient. RNA's isolated frora virions and from purified nucleocapsid are indistinguishable in sedimentation behavior. The sedimentation coefficient of SV5 RNA was estimated to be 50 S in sucrose gradients containing 0.05 M NaCl. On the basis of its ribonuclease sensitivity, base composition, and sedimentation behavior, SV5 RNA appears to be single-stranded.

The methods used in the studies of SV5 were applied to pneumonia virus of mice (PVM), an unclassified virus whose structure had not been previously determined. PVM virions are spheres 80-120 m\i in diameter, or filaments of similar diameter with lengths up to 3 |i. The particles possess an outer, spike-covered envelope and helical internal component 120-150 A in diameter. Virus particles acquire their envelope by a budding process at the cell membrane; mature particles are seen only extracellularly. Dense inclusions are prominent in the cytoplasm of PVM-infected BHK21 cells and appear to consist of aggregates of the PVM internal component. The helical component was isolated in a cesium chloride gradient from extracts of osmotically shocked cells. Murine erythrocytes, which are agglutinated by PVM, adsorb to the surfaces of infected cells and to budding and extracellular PVM particles. On the basis of its structure and morphogenesis, PVM appears to be a myxovirus; however, the details of its structure and replication differ frora either of the two established subgroups of myxoviruses and suggests that a third subgroup of these viruses exists.

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