Replication of semliki forest virus: three forms of viral RNA produced during infection.

Abstract

The mechanism for the replication of animal RNA viruses has been the subject of intense investigation. With few exceptions (Gomatos and Tamm, 1963; Sonnabend et al., 1964), however, these studies have been on picornaviruses, specifically poliovirus (Baltimore, Becker, and Darnell, 1964) or members of the encephalomyo-(arditis group (Montagnier and Saunders, 1963). This report will present studies on the replication pattern of Semliki Forest virus, a group A arbovirus. Semliki Forest virus has some properties advantageous to the study of animal virus replication, including a particle-to-infectious-virus ratio close to 1 (Cheng, 1961), wide host range, rapid growth to high titers in tissue culture, and little or no pathogenicity for man (Andrews, 1964). In this paper the forms of viral RNA produced in the cytoplasm of infected chick embryo fibroblasts are considered. Materials and Methods.-Virus: The Kumba strain of Semliki Forest virus (SFV) employed in these studies was originally obtained from Dr. Alick Isaccs, Mill Hill, London. Pools of virus were prepared by infection of actinomycin D (ACM)-treated (0.5 ,g/ml) chick embryo fibroblasts with low multiplicities of virus. Virus was grown in Eagle's medium, harvested after 14 hr by the methods of Taylor (1965), and assayed by a plaque titration method (Friedman, 1964). Radioactive virus was grown in chick cells treated with ACM (0.5 ,gg/ml) in the presence of H3uridine and H3-adenosine. Fluids were harvested after 18 hr and virus was purified by the method of Cheng (1961) with the addition of a final dialysis to remove small tritiated products. The specific activity of the final preparation was usually about 1 cpm/2000 infectious units. The RNA extracted from this virus preparation by the method of Cheng (1958) had a sedimentation value of 42S and was ribonuclease-sensitive. Sedimentation velocities of the tritiated virus performed on sucrose gradients indicated a peak of radioactivity at about 350S which corresponded to the peak of virus infectivity (cf. Cheng, 1961). Radioactivity and infectivity were also present in the pellet, possibly due to viral aggregation. Cells: One-day-old primary chick embryo monolayers (CE) were prepared in 100-mm plastic Petri plates by previously described methods (Taylor, 1965); these cells were employed in all studies. When confluent, these monolayers contained about 2.5 X 107 cells/plate. Pulse-labeling and viral RNA extraction: Pulse-labeling was carried out by adding to the cell cultures 1 ml of tissue culture fluid containing the isotope. After an appropriate pulse period, the monolayers were washed five times with cold phosphate buffered (0.01 M, pH = 7.2) 0.85% saline and once with potassium phosphate buffer (0.1 M, pH = 7.1). Cells were removed from plastic Petri dishes with a rubber stopper and suspended in 1 ml of potassium phosphate buffer. The cells were disrupted with a Dounce homogenizer, and the homogenate was centrifuged at 600 g for 10 min. The remaining cytoplasmic fraction was extracted with 5% sodium dodecyl

The Intracellular Site and Sequence of Sindbis Virus Replication

“…It has been demonstrated that arbovirus RNA synthesis in infected chick embryo fibroblasts resembles in principle that of picornaviruses (Sonnabend et aL I964;Friedman, Levy & Carter, 1966 ). The present study deals with the intracytoplasmio localization in infected chick embryo fibroblasts of Sindbis virus RNA, and with the time course of synthesis of the different viral RNA species, coat proteins and virions.…”

Stability and Precursor Relationships of Virus RNA

“…Pulse experiments with [~H]-uridine demonstrated that in Semliki Forest-infected cells a partially RNase-resistant 2o S RNA appears first and was followed by RNA of ~6 S and 42 S (Friedman, Levy & Carter, 1966;Cartwright & Burke, 197o). Thus it was possible that these components were in precursor-product relationship.…”

Polyadenylic Acid Sequences in the Virus RNA Species of Cells Infected with Semliki Forest Virus

“…In this paper we use several different procedures to examine the virus-specific RNA from cells infected with Semliki Forest virus, an alphavirus, for the presence of poly A tracts. Such viruses are of interest because the virus particle contains a molecule of single-stranded RNA which is infectious (Friedman, Levy & Carter, 1966) and which therefore is presumed to act as a messenger for its own RNA replicase. In addition, the infected cell contains three other single-stranded RNAs of the same polarity, two of which have been shown to be associated with polysomes (Kennedy, i97z;Moshowitz, I973;Rosemond & Sreevalsan, 1973) and are therefore putative messengers.…”