Student Theses and Dissertations
Date of Award
1971
Document Type
Thesis
Degree Name
Doctor of Philosophy (PhD)
Thesis Advisor
Lawrence Caliguiri
Keywords
poliovirus, intracellular membranes, viral RNA synthesis, smooth microsomes, choline metabolism, guanidine inhibition
Abstract
Picornavirus multiplication is intimately associated with intracellular membranes and affects their production and distribution within the cell. We have shown that the density distribution of cytoplasmic membranes separated by isopycnic centrifugation in discontinuous sucrose gradients is progressively altered after infection of HeLa cells with poliovirus. The most striking change is a very large increase in the smooth microsomal membranes with which viral RNA synthesis is associated. These membranes begin to increase between 2.5 and 3.5 hours after infection, and continue to proliferate late in the virus growth cycle, at a time when cellular protein and nucleic acid syntheses are much reduced. These smooth membranes differ from the membranes in the corresponding fraction from uninfected cells in several ways. They have a higher phospholipid/protein ratio and the phospholipid composition is altered. We have confirmed that the viral RNA polymerase activity is firmly associated with the membranes in this fraction and that it may be localized in a complex with a sedimentation constant of 130 S after lysis of the membranes with deoxycholate. We have utilized 3H-choline as a specific precursor of choline-containing phospholipids, to study the synthesis of cytoplasmic membranes. We have confirmed that the incorporation of 3H-choline into cytoplasmic membranes is greatly stimulated by poliovirus infection. When uninfected or infected cells are pulsed for 3 minutes with 3H-choline, and then fractionated on discontinuous sucrose gradients, the highest activity is found in the rough microsomal membranes which form a thick pellicle in the lower third of the centrifuge tube. These membranes probably constitute the intracellular location for the synthesis and incorporation of phospholipids into membranes. Uninfected cells still show this distribution of 3H-choline after 30 minutes of labeling, but in infected cells, a bimodal distribution is observed. The radioactivity is preferentially incorporated into the accumulating smooth microsomal membranes as well as into the pellicle of rough microsomal membranes. When infected and uninfected cells are pulse-labeled for 3 minutes with tritiated glycerol, the infected cells incorporate 4-5 times as much radioactivity as uninfected cells. About 80% of the label is incorporated into the rough endoplasmic reticulum. After a 60-minute chase period, the distribution of radioactivity does not change significantly in uninfected cells. By contrast, in infected cells, much of the label shifts from the rough microsomes into the smooth membranes, particularly those with which viral RNA synthesis is associated. Thus, it appears that there is preferential synthesis in the rough endoplasmic reticulum of lipid destined to become part of the smooth membranes which accumulate throughout infection. Treatment of infected cells with 1.0 to 2.0 mM guanidine-HCl inhibits viral RNA synthesis, but does not measurably affect cellular metabolism. We have confirmed the observation that early guanidine treatment of cells infected with at least 50 PFU per cell of poliovirus does not block the virus-induced stimulation of choline incorporation, although this stimulation is diminished. However, such guanidine-treated, infected cells do not produce the large quantities of smooth surfaced membranes which normally accumulate in infected cells. Indeed, the density distribution of membranes in guanidine-treated, infected cells resembles closely that of uninfected cells. This suggests that poliovirus products may affect the control of cellular membrane synthesis in two separable ways: incorporation of precursors, and the conversion of rough to smooth-surfaced membranes. We have also studied the choline-mediated blocking of the inhibitory action of guanidine on poliovirus biosynthesis in three cell types. Poliovirus biosynthesis shows increasing sensitivity to inhibition by guanidine in the different cells in the order: HeLa cells, LLC-MK2 cells, and primary rhesus monkey kidney cells. When viral replication is more strongly inhibited in a given cell type, a higher concentration of choline is required to block the inhibition. Kinetic studies in all three cell types show that 10 mM choline rapidly reverses the inhibitory effects of 0.4 mM guanidine. After addition of choline, viral multiplication proceeds at a rate indistinguishable from that in control cells that have received neither guanidine nor choline. Choline does not appear to enhance the rate of exit of 14C-guanidine from cells incubated in guanidine-free medium. A variety of amino acids and compounds structurally related to choline are also known to have the ability to block guanidine inhibition of poliovirus replication. Although choline and methionine act synergistically in blocking the virus-inhibitory action of guanidine, choline and dimethylethanolamine are incapable of synergism. This suggests that guanidine antagonists may be grouped into several classes. Choline, dimethylethanolamine and methionine all suppress the growth-supporting activity of 0.4 mM guanidine on a guanidine-dependent strain of poliovirus type 1. Possible mechanisms by which choline and other guanidine antagonists might block the action of guanidine on poliovirus multiplication are considered, and the implications of the results of our studies for these mechanisms are discussed.
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Recommended Citation
Mosser, Anne G., "Poliovirus Replication: Interactions with Cytoplasmic Membranes and with Compounds Which Affect Virus Biosynthesis" (1971). Student Theses and Dissertations. 551.
https://digitalcommons.rockefeller.edu/student_theses_and_dissertations/551
Comments
A thesis presented to the faculty of The Rockefeller University in partial fulfillment of the requirements for the degree of Doctor of Philosophy