FIG 4.

Helper activity of L-A variants. (A) Diagram of a cytoduction experiment to produce strain 1149, which maintains M2 virus by L-A proteins expressed from a vector. L-A-2 and M2 viruses (shaded and open hexagons, respectively) from strain 1137 were transferred to strain 2928 carrying plasmid p1290 (indicated by open circles in the nucleus). Although the cytoductants originally contained L-A-2 and M2, L-A-2 was eliminated by overexpression of viral proteins from the plasmid. (B) (Top) RNAs from two cytoductants (lanes 2 and 3) were analyzed in an agarose gel. RNAs from donor strain 1137 were used as a control (lane 1). Note that both cytoductants have lost L-A-2 but maintain large amounts of M2 dsRNA, visible as discrete bands in the ethidium bromide (EtBr)-stained gel (lanes 2 and 3). (Center) L-A-2 is detected by hybridization with an L-A-2-specific probe. (Bottom) K2 killer activity of isolated cytoductants. (C) Diagram of cytoduction experiments to introduce M2 virus from donor strain 1149 into three K-o recipient strains carrying L-A-lus or L-A helper viruses. The helper viruses (of any type) are indicated by shaded hexagons and M2 virus by open hexagons. (D) Killer assays of isolated cytoductants in each case. The recipient strain name is given below each image, and the respective helper virus is identified at the top. Only if an L-A variant has helper activity for M2 are the cytoductants K2 killers (+); otherwise they are nonkillers (−). (E) M2 maintenance by L-A or L-A-lus in ski mutants. M2 virions from strain 1161, which are maintained by viral proteins expressed from vector pI2L2, were introduced by cytoplasmic mixing into five strains carrying L-A: strain BY4741 (wild type [WT]) (lanes 1 to 3) or mutant derivative strains with deletions of SKI2 (strain Y05307) (lanes 4 to 6), SKI3 (strain Y05604) (lanes 7 to 9), SKI1 (strain Y04540) (lanes 10 to 12), or SKI7 (strain Y01852) (lanes 13 to 15). M2 virions from strain 1161 were also introduced into the L-A-lus-containing strain 1094, which carries the ski2-2 mutation (lanes 16 to 18). In each case, RNAs from the recipient strain and two independent cytoductants (Cytod.) were separated on an agarose gel and were analyzed by Northern hybridization with probes specific for L-A (or L-A-lus) and M2. All cytoductants carried 23S RNA (indicated by the asterisk), present in the donor strain but absent in the recipients. At the bottom, isolated colonies of independent cytoductants from each strain are analyzed for K2 killer activity.