Association of the polioviral RNA polymerase complex with phospholipid membranes

Abstract

Polioviral RNA polymerase complex, which consists of enzyme, template, and nascent RNA, is membrane bound in vivo. The solubilized RNA polymerase complex associated spontaneously in vitro with phospholipid bilayer membranes (liposomes) of defined composition. The degree of association at 37°C was greater for those membranes that were more fluid, suggesting that the binding involves the interaction of the RNA polymerase complex with the hydrocarbon chains in the interior of the lipid bilayer. The polymerase activity was not enhanced by addition of the lipid; in fact, the addition of some of the longer-chain lipids resulted in up to a 40% inhibition of the polymerase activity. Spin-label electron paramagnetic resonance experiments, which measured the membrane fluidity, and kinetic experiments on the rate of incorporation of tritiated UTP into RNA by the polymerase were performed as a function of temperature. The results indicated that the activity of the polymerase was not affected by the physical state of the phospholipid membrane and that its active site was not intimately associated with the membrane. Analysis of both the viral and host polypeptides associated with the smooth membrane-bound polymerase indicated that X was the primary viral polypeptide present. In addition, host polypeptides of molecular weight 86,000, 62,000, 54,000, and 46,000 were also present. If the membrane was disrupted with detergent, polypeptide X was released from the polymerase activity, suggesting that X may play a role in binding the polymerase to the membrane. In an analogous manner, polypeptide X associated spontaneously with phospholipid membranes to a greater extent than the capsid polypeptides. Analysis of both the host and viral polypeptides associated with the viral RNA polymerase purified by precipitation in 2 M LiCl indicated that host polypeptides

Isolation of poliovirus 2C mutants defective in viral RNA synthesis

“…Previous data suggested that protein 2C is involved in RNA synthesis. The high degree of amino acid sequence conservation of protein 2C among picornaviruses (1,10,23) argues for its importance, and the demonstration that 2C is associated with the membrane-bound viral replication complexes in poliovirus-infected cells (5,6,33) suggests that it is inherent in the replication process. This perspective is further supported by the observation that several guanidineresistant and guanidine-dependent poliovirus mutants map to within the region that encodes protein 2C (15,(23)(24)(25).…”

“…" All single and mixed infections were carried out at 39.5°C for 4 h with a multiplicity of infection of 5 for each virus 6. Titers of the viruses harvested from single or mixed infections containing 106 HeLa cells each in 2 ml of minimal essential medium.…”

RNA recombination in animal and plant viruses.

“…In retroviruses, this is made possible by a physical linkage of two RNA templates in heterozygotes, thus facilitating template switching (18). In most RNA viruses, RNA synthesis occurs in membraneassociated compartments, thus allowing a local concentration of RNA molecules (11,16,23,62). It also has been proposed that the complementary sequence in the secondary structure of RNA could allow two different RNA molecules to form a heteroduplex (14,56,87).…”

A human virus protein, poliovirus protein 2BC, induces membrane proliferation and blocks the exocytic pathway in the yeast Saccharomyces cerevisiae.

“…panels). All three proteins 2B, 2C and 2BC seem to interact with membranes in mammalian cells (Butterworth et al, 1976;Bienz et al, 1983;Tershak, 1984). To test this possibility in yeast cells, we employed standard differential centrifugation techniques to fractionate yeast lysates from spheroplasts (Bankaitis et al, 1989).…”

“…With regard to the generation of these membranous structures and the viral proteins involved in this process, it was found that apart from the viral RNA polymerase, 3DP°1, other viral non-structural proteins were tightly associated with the poliovirus replication complexes (Girard and Baltimore, 1967;Butterworth et al, 1976;Bienz et al, 1983;Tershak, 1984). Of interest in this regard was the association of 2BC and 2C not only with the membrane vesicles, but also with the viral nucleic acids (Butterworth et al, 1976;Bienz et al, 1983;Tershak, 1984). Initial studies localized several poliovirus proteins in these membranous vesicles and suggested that the precursor 2BC could play an important part in the generation of the vesicles (Bienz et al, 1983).…”