Serial passaging of Semliki Forest virus in BHK cells at a constant input multiplicity of 50 p.f.u./cell resulted in a 4 log 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 CsCl resolved the DI particles (identified by interference analyses) from standard virus. The buoyant density of the DI particles (1.23 g/ml) was higher than that of standard virus (ρ = 1·20 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 × 10), DI particles contained two smaller pieces of RNA of mol. wt. 0.81 and 0·75 × 10 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 0·02 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.


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