Table 1.

Quantitation of RNA species produced in transfected cells programmed with plasmid encoding a minigenome bearing one of various trailer mutationsa

Trailer mutantT7 transcriptbEncapsidated genome (protected from nuclease)cTotal positive-sense RNA, NPLRelative genome template activityd
0eNPΔLfNPLg
wt37,700h (1.0)i,100 j 1,288,h 3.4 j 3,757,h 10 j 19,257,h 51 j (5.1)k 76,451h (1.0)l 3.97 (1.0)m
1C17,600 (0.46), 100 0, 0 4,076,23 5,252,30 (1.3)13,760 (0.18)2.62 (0.66)
2G132,400 (3.5), 100 576,0.44 22,118, 17 18,07114 (0.82)57,569 (0.75)3.19 (0.80)
3A45,900 (1.3), 100 2,006,4.4 18,263, 40 13,579,30 (0.74)24,818 (0.32)1.83 (0.47)
4G44,000 (1.2), 100 4,017,9.1 17,776, 40 30,217,69 (1.7)100,394 (1.3)3.32 (0.84)
4C11,400 (0.30), 100 1,242,11 3,577, 31 11,715,103 (3.3)44,409 (0.58)3.79 (0.95)
5A33,000 (0.88), 100 1,564,4.7 9,223, 28 11,711,35 (1.3)37,714 (0.49)3.22 (0.81)
6C49,800 (1.3), 100 1,415,2.8 20,368, 41 11,120,22 (0.55)45,214 (0.59)4.07 (1.03)
7C51,900 (1.4), 100 1,411,2.7 9,608, 19 8,444,16 (0.88)16,290 (0.21)1.93 (0.49)
  • a Data from the representative experiment shown in Fig. 3.

  • b Amount of plasmid-derived T7 transcript with uncleaved ribozyme, averaged from the three support conditions 0, NPΔL, and NPL (e.g., for wt, Fig. 3A, lanes 1, 2, and 3). This is taken as a surrogate measure of the amount of total T7-synthesized minigenome (see the text).

  • c Amount of minigenome remaining after micrococcal nuclease treatment under the indicated support conditions: 0, NPΔL, and NPL (e.g., for wt, Fig. 3B, lanes 1, 2, and 3).

  • d The relative activity of each minigenome as template for the synthesis of positive-sense RNA was determined by dividing the total amount of positive-sense RNA by the total amount of encapsidated negative-sense RNA (NPL) for that minigenome. Since the values for positive sense versus negative sense are from separate blots, the quotient is an arbitrary unit. Compared between minigenomes, it gives an indication of relative template activity.

  • e Residual undigested minigenome, reflecting a background due to incomplete and variable nuclease digestion in the crude cell lysates.

  • f Reflects encapsidation by N and P in the absence of active RSV polymerase.

  • g Reflects genome synthesized and encapsidated in the presence of active RSV polymerase.

  • h Phosphorimager signal units.

  • i Values in parentheses are amount of T7 transcript relative to wt as 1.0.

  • j The numbers in boldface are values normalized to the T7 transcript value for that minigenome as 100.

  • k Encapsidation value relative to NPΔL for that minigenome as 1.0 is given in parentheses. Values of >1 indicate genome amplification by the RSV polymerase. Values of <1.5 are considered marginal.

  • l Values in parentheses are relative to wt as 1.0.

  • m Values in parentheses are relative to wt as 1.0.