Molecular Weight of Poliovirus Ribonucleic Acid

Nicole Granboulan, Marc Girard
1969 Journal of Virology  
Purified poliovirus singleand double-stranded ribonucleic acids (RNA) were examined by electron microscopy. The length of both molecules was found to be 2.37 ,um. The uncorrected sedimentation coefficient for single-stranded RNA is 335, as compared to 275 for the RNA of tobacco mosaic virus. It is calculated from these results that the molecular weight of the sodium form of poliovirus is 2.6 X 106 daltons. In contrast to extensive studies of the molecular weight of tobacco mosaic virus (TMV)
more » ... onucleic acid [RNA (6, 7)], only limited physical data exist for the RNA molecules of animal viruses. Poliovirus RNA has been claimed to behave similarly to TMV RNA in the analytical ultracentrifuge (25); it has, therefore, been assumed that its molecular weight was approximately 2 X 106. Measurement of the lengths of both singleand double-stranded RNA forms under the electron microscope did not corroborate this figure and led to a new estimation of the molecular weight of poliovirus RNA, which was in turn verified by sedimentation analysis. MATERIALS AND METHODS The Mahoney strain of poliovirus was grown in HeLa cells as described previously (4). The cells were washed with ice cold Earle's saline solution 7 hr after infection (10). Cytoplasmic extracts were prepared (12) and treated with 1% sodium dodecyl sulfate (SDS). These were centrifuged for 4 hr at 75,000 X g. The sedimented virus was suspended in SDS buffer [0.01 M tris(hydroxymethyl)aminomethane (Tris)-hydrochloride (pH 7.4), 0.1 M NaCl, 0.001 M ethylenediaminetetraacetate (EDTA), 0.5% SDS] and purified by centrifugation through a 28-ml gradient of 15 to 30% (w/w) sucrose in SDS buffer (Spinco model L2, SW 25.1 rotor, 3 hr at 63,000 X g, 22 C). Viral RNA was extracted according to Mandel (22) by treatment of the purified virus with SDS at pH 4.1. SDS was then precipitated by addition of 0.1 M KCl in the cold. Viral RNA was further purified by centrifugation through a 28-mil gradient of 15 to 30% sucrose in 0.01 M Tris-hydrochloride (pH 7.4), 0.1 M KCJ, 0.001 M EDTA (Spinco model L2, SW 25.1 rotor, 18 hr at 60,500 X g, 5 C). Purified poliovirus RNA was then dialyzed against 0.02 M Tiis-hydrochloride (pH 7.5) concentrated by dialysis against Ficoll (Pharmacia), and stored frozen in liquid nitrogen. TMV grown in 14CO2 was a generous gift from L. Hirth. The virus was treated with 20,ug of Pronase per ml (Calbiochem) for 40 min at 22 C, then mixed with an SDS-treated cytoplasmic extract from 101 HeLa cells. The mixture was extracted with phenol at 37 C. After precipitation with ethyl alcohol, the labeled RNA was further purified by sucrose gradient centrifugation in SDS buffer. Poliovirus double-stranded RNA was prepared from HeLa cells which had been labeled with 20 ,uc/ml of tritiated uridine (22 c/mmole) in the presence of 5 ,g of actinomycin D per ml. At 3.8 hr after infection, cytoplasmic extracts were prepared, and centrifuged for 40 min at 100,000 X g at 4 C. Resulting pellets were suspended in SDS buffer and precipitated with 2 M LiCl. Labeled double-stranded RNA was purified from the LiCl supernatant fractions by exclusion chromatography on 2% agarose (Sepharose 2 B, Pharmacia) according to Baltimore (3). Samples for electron microscopy were prepared following the technique of Kleinschmidt (17, 18). Single-stranded RNA was first diluted with 8 M urea (15, 16) . Double-stranded RNA was examined with and without addition of 8 M urea. Approximately 0.2 ml of a 1 M ammonium acetate solution, pH 8, containing from 1.7 to 4.0 Lg of RNA per ml and 0.01% cytochrome c was allowed to flow down a clean inclined glass slide onto a hypophase of either 0.015 M ammonium acetate or 4 M urea. The resultant film was picked up on carbon coated Formvar films supported by 300 mesh grids. The specimens were dried in ethyl alcohol and shadowed with uranium oxide at an angle of 7°. Electron micrographs were made with a Philips EM 200 at a nominal magnification of 9,100 to 15,600 times; actual magnifications were determined and controlled with carbon grating replicas. Lengths of RNA molecules were determined on positive prints with a map measurer. As previously reported (16), grids rich in double-stranded RNA were more easily obtained than grids rich in single-stranded RNA. RESULTS AND DISCUSSION All poliovirus RNA molecules observed were linear, i.e., neither circular nor branched (Fig. la) . 475 VoL 4, No. 4
doi:10.1128/jvi.4.4.475-479.1969 fatcat:pc2tjmt2k5ezxc546ntjcoxaxi