IN-VITRO PACKAGING OF THE SINGLE-STRANDED RNA GENOMIC PRECURSORS OF THE SEGMENTED DOUBLE-STRANDED-RNA BACTERIOPHAGE-PHI-6 - THE 3 SEGMENTS MODULATE EACH OTHERS PACKAGING EFFICIENCY

Bacteriophage phi 6 is a double-stranded RNA (dsRNA) virus that has a genome composed of three linear dsRNA segments (I, m, s). These are encapsidated into a dodecahedral procapsid particle consisting of proteins P1, P2, P4 and P7. Expression of the cDNA copy of the L segment in Escherichia coli leads to the formation of empty procapsid particles. These particles are able to package the plus-sense single-stranded RNA (ssRNA)s of each genome segment in vitro. We have used this in vitro system for a detailed study of phi 6 RNA packaging. The reaction conditions for RNA packaging were optimized using a RNase protection assay. The RNA packaging reaction is dependent on divalent cations (either Mg2+ or Mn2+) and requires a nucleoside triphosphate (NTP) as an energy source. Any one of the rNTPs, dNTPs or ddNTPs can support the RNA packaging. Purine nucleotides support packaging better than pyrimidine nucleotides, GTP being preferred to ATP. The plus-sense ssRNA of each the three genome segments can be packaged independently into the procapsid. However, when two or three segments are packaged simultaneously, regulatory effects modulating the packaging efficiency can be detected between the segments. The packaging of the s and In segments is more efficient when they are packaged alone, compared to a situation in which they are packaged with the other segments. In contrast, the packaging of the I segment is very inefficient alone, but is enhanced when packaged together with the m segment. We propose that each segment has a preferred high-affinity binding site in the procapsid particle and packaging of the in segment creates the high-affinity binding site for the I segment. If any of the segments is missing from the packaging reaction the other segments can occupy its binding site.