TABLE 2.

Virion incorporation efficiencies of vRNA segments^a

NP	No. of VLPs possessing at least one vRNA (per ml)b	No. of VLPs possessing at least four vRNAs (per ml)c
Wild type	31,900	17,800
D72A	450	0
R74A	62,500	390
K113A	2,160	0
R156A	167,000	11
R174A	8,980	0
R175A	100,000	0
R195A	26,200	0
R199A	51,000	0
K325A	5,700	0
R361A	21,080	0

^a293T cells were transfected with eight PolI constructs for eight RNA segments [i.e., PB1, PB2, PA, NA, M, NS, HA-GFP, and NP(Met⁻), which lacks a start codon] and five protein expression constructs for PB1, PB2, PA, HA, and mutant or wild-type NP. Forty-eight hours later, VLPs were harvested, treated with TPCK trypsin, and used to determine the virion incorporation efficiencies of viral RNA segments as described in notes b and c. The results shown are representative data from three independent experiments.

^bVLPs and WSN helper virus were used to infect MDCK cells. With helper virus providing polymerase proteins and wild-type NP, GFP-positive MDCK cells represented VLPs that possessed at least one vRNA, i.e., HA-GFP vRNA (Fig. 3).

^cInfectious VLPs were used to infect MDCK-NP cells. In this approach, GFP expression from HA-GFP vRNA requires the expression of the PB2, PB1, and PA proteins from the respective vRNA segments. GFP-positive MDCK cells therefore represented VLPs that contained at least four vRNAs (i.e., the HA-GFP, PB1, PB2, and PA vRNAs).