We have studied the effect of the drug pactamycin on protein synthesis in poliovirus-infected HeLa cells. At a concentration which primarily inhibits initiation of protein synthesis, the spectrum of poliovirus proteins synthesized is markedly changed. The amount of NCVP 1, the capsid precursor, is greatly reduced relative to NCVP 2 and the amount of NCVP X is slightly reduced. Since it is believed that there is only one major site for the initiation of protein synthesis on the poliovirus genome, we interpret this differential effect on the poliovirus proteins to be an indication of their relative distance from the initiation site. On this basis, we propose a gene order for the poliovirus genome (5'-* 3') of NCVP 1, NCVP X, NCVP 2.

We present evidence that poliovirus can be encapsulated in synthetic large phospholipid vesicles. The virus associated with the vesicles is found to be (i) resistant to antiserum against poliovirus and (ii) infectious for cells that are normally resistant to virus infection because of a membrane restriction. Our interpretation of these results is that the virus is entrapped in the interior aqueous space of the vesicles and that this vesicle-associated virus is introduced directly into the cytoplasm of the cells via fusion of the vesicles with the cellular plasma membrane, bypassing the surface receptor-mediated restriction.

An RNA sedimenting at 10 S with a molecular weight of 2.3 X 106, was isolated from rabbit reticylocyte polyribosomes. When this RNA is added to a cell-free extract from Krebs II ascites cells, both a and ,B chains of rabbit hemoglobin are synthesized; g chains are made in about 50% excess over a chains.Isolation and characterization of the messenger RNA for a and # chains of rabbit hemoglobin has been reported by several groups (1-7). The informational content of this RNA has been assayed by adding it to a eukaryotic cell-free extract that was already synthesizing a homologous hemoglobin (3, 7) or myosin (5), or to a bacterial cell-free extract (4). The protein made in these extracts was analyzed by separation of the polypeptide chains into those whose synthesis was directed by endogenous messenger RNA and those whose synthesis was the result of added RNA. We report here the synthesis of both a and 13 chains of rabbit hemoglobin when 10S messenger RNA from rabbit reticulocytes is added to a cell-free extract from Krebs II ascites cells. This cell-free extract can be incubated to eliminate the synthesis of endogenous protein, while it retains the ability to respond to added viral RNA (8,9). Our results extend the use of previously incubated extracts of Krebs II ascites cells to characterization of proteins encoded by isolated mammalian messenger RNAs. METHODSThe 10S messenger RNA was prepared from rabbit reticulocytes by the method of Labrie (2). Polyribosomes were dissociated in EDTA, and the ribonucleoprotein particle that contained 10S messenger RNA was separated from ribosomal subunits on a sucrose density gradient (2). The RNA was then precipitated by adjusting the appropriate fractions to 0.1 M NaCl and 0.5% sodium dodecyl sulfate (SDS), followed by the addition of 3 volumes of ethanol. After 2 hr at -20'C, the precipitate was collected by centrifugation, dissolved in 0.1 M NaCl-0.5% SDS-1 mM EDTA-0.01 M Tris [pH 7.4 (SDS buffer)], layered over a 15-30% sucrose density gradient made in the same buffer, and centrifuged for 21 hr at 22°C and 27,000 rpm in the Beckman SW 27 rotor. The gradient was monitored for Aseonm; fractions containing RNA sedimenting at 10 S were pooled and precipitated with ethanol.The precipitated RNA was dissolved in SDS buffer and extracted with phenol-chloroform, as described by Penman (10). After precipitation with ethanol, the RNA was washed at least three times with ethanol at -20°C to remove SDS, dissolved in water at a concentration of 1 mg/ml, and stored 2716 at -70°C. 40 ug of purified 10S RNA was prepared from 10 mg of polyribosomes. Maintenance of Krebs II ascites cells in mice has been described (8). A cell-free extract was prepared from these cells according to a method communicated to us by M. B. Matthews, and used by him and others (8, 9). Mice bearing tumors were killed 7-10 days after inoculation, and the ascites cells were removed from the peritoneal cavity into 0.146 M NaCl-35 mM Tris (pH 7.5). They were collected by centrifugation at 800 rpm (120 X g) for 2 ...

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We obtained a persistently infected line of Chinese hamster ovary cells by selection for resistance to reovirus infection. The cells were persistently infected by a population of viruses that were (i) cytopathic for parental chinese hamster ovary cells and (ii) similar to wild-type reovirus in molecular characteristics. The growth rate, plating efficiency, and morphology of the cells were altered. A large majority of the cells in the population were infected. There was no detectable interferon present in the medium. The cells were relatively resistant to a wide range of viruses. Cells can acquire resistance to virus infection by a number of mechanisms. The least understood of these are mechanisms that do not involve extracellular factors such as interferon and humoral antibodies (15, 24). To study mechanisms of intracellular resistance we have selected Chinese hamster ovary (CHO) cells that are resistant to reovirus. In this communication, we describe one such cell line (VRR) that is characterized by the constant presence of infectious reovirus in the media. Such persistent infections are well known in virology, primarily with viruses that are weakly cytopathic such as certain enveloped viruses (2, 5, 14, 17, 19, 22-24). The molecular nature of such infections is poorly understood. We believe that this cell line is a unique manifestation of a reovirus infection and that the further study of such an infection with a virus whose molecular biology is extremely well characterized (11) should yield important information regarding the general phenomena of persistence. MATERIALS AND METHODS Cells. The parental cell line was CHO-KI (CCL 61) obtained from the American Type Culture Collection and periodically cloned (10). The cells are routinely maintained in Ham F-12 medium (6) buffered with 25 mM N-2-hydroxymethylpiperazine-N'2-ethanesulfonic acid (with a consequent reduction in NaH2CO, to maintain isotonicity) (Grand Island Biological Co.). This medium was supplemented with 10% fetal calf serum (Microbiological Associates). The cells are grown at 37 C in Forma water-jacketed incubators. Both CHO and VRR cells were periodically assayed for mycoplasma contamination by L. Hayflick.