Defective-Interfering Particles of Semliki Forest Virus: Structural Differences between Standard Virus and Defective-Interfering Particles

Abstract

SUMMARYSerial passaging of Semliki Forest virus in BHK cells at a constant input multiplicity of 5o p.f.u./cell resulted in a 4 log10 drop in the yield of infectious virus by passage 9. An interference analysis showed that this drop was due to the presence of defective-interfering (DI) particles. Attempts were made to separate the DI particles from standard virus by equilibrium and velocity centrifugation. Only equilibrium centrifugation on CsC1 resolved the DI particles (identified by interference analyses) from standard virus. The buoyant density of the DI particles (I.23 g/ml) was higher than that of standard virus (p = I'2o g/ml).No difference was observed between the structural proteins of standard virus and DI particles. Analysis of the RNA of standard virus and DI particles showed that whereas standard virus contained only 42S RNA (mol. wt. approx, 4.2 × lo6), DI particles contained two smaller pieces of RNA of mol. wt. o.8I and 0"75 × Io 6 respectively. Infectivity assays showed that these low mol. wt. species were not only non-infectious but also interfered with the infectivity of 42S RNA from standard virus. Nucleocapsids derived from purified DI particles had a buoyant density o.o2 g/ml greater than the nucleocapsids from standard virus. Analysis of the RNA from DI nucleocapsids showed it to be entirely of the low mol. wt. class.To account therefore for the density difference not only between DI particles and standard virus but also between their respective nucleocapsids we propose that each SFV DI particle contains several molecules of the low mol. wt. RNA species.

Assay of Defective-interfering Semliki Forest Virus by the Inhibition of Synthesis of Virus-specified RNAs

“…wt. (0.8 x 106) ofDI RNA of the same virus at passage 8 (Bruton & Kennedy, 1976), suggesting that the entire genome is required for interference.…”

“…This has to be compared with the six high m.o.i. passages of SFV, also in BHK cells, which were required before Bruton & Kennedy (1976) had evidence of DI virus production from the decreased yield of infectious virus. It also emphasizes the importance of prolonged incubation in the generation of DI virus noted for Sindbis virus (King et aL, 1979).…”

“…M0nolayers were incubated at 33 °C for 18 h and the tissue culture fluids (TCF) were then clarified by low-speed centrifugation before storage at -70 °C. DI SFV was prepared by serial passage in BHK cells at an m.o.i, of 50 according to Bruton & Kennedy (1976), and this was the direct descendant of the passage 8 virus used by Dimmock & Kennedy (1978). In this report DI SFV stocks of passages 12 and 13 were used except where noted.…”

“…Defective-interfering (DI) virus particles accumulate during serial passage of many animal viruses at a high multiplicity of infection (Huang & Baltimore, 1970, 1977, including the alphaviruses Semliki Forest virus (SFV) and Sindbis virus (Bruton & Kennedy, 1976;Schlesinger et al, 1972). All DI viruses have a portion of their genome deleted and can only replicate in the presence of standard (infectious) virus and this interferes with the multiplication of the standard virus.…”

Semliki Forest Virus-induced, Immune-mediated Demyelination: Adoptive Transfer Studies and Viral Persistence in Nude Mice

“…An ELISA based on that of Voller et al (1976) was used to measure anti-SFV IgG levels. Virus was purified by ultrafiltration and sucrose gradient centrifugation as described by Bruton & Kennedy (1976). A single sucrose gradient was run and the buffer used throughout was 0.05 M-Tris, 0.1 M-NaC1, 0-1 mM-EDTA, 200 mu-glycine, pH 7.4, containing no foetal calf serum (FCS).…”