Due to the multiple nucleotide substitutions of the SQL mutation, the observed 
reversion frequency of 10"* is most likely due to a single-nucleotide, second-site 
mutation, rather than reversion of the SQL mutation itself. 
5. PROPERTIES OF SFV/HELPER 2 VIRUS 
An SFV helper virus containing the SQL mutation should provide a much higher 
level of biosafety than use of either SFV/Helper 1 or SFV-SQL alone. Thus 
Liljestrom and colleagues transferred the SQL mutation into the Helper 1 
plasmid, creating the SFV/Helper 2 expression system. 
The efficiency of the new helper was tested in packaging reactions using SFV3- 
lacZ as the recombinant RNA. Compared to the wild type SFV (SFV4), SFV3- 
lacZ/Helper 2 generated similar levels of viral protein 6.5 hours post-transfection 
and similar rates of assembly of pulse-lableled spike protein into mature virus 
particles (data not shown). 
a. Leakiness of SFV/Helper 2 Virus 
The residual infectivity of SFV-lacZ/Helper 2 particles, or "leakiness," was 
measured by exposing BHK cells to unactivated particles at various multiplicities 
of infection and scoring for LacZ-positive cells. Table 5 shows the result of 
testing four different stocks of virus. Only about one in 3x10 s particles is capable 
of entering BHK cells under these conditions. 
(The mechanisms causing leakiness are unknown, but may result from distortion 
or cleavage of some of the spike proteins, not all of which need to be activated 
for infectivity (Salminen et al., 1992). Such mechanisms are distinct from genetic 
reversion, primary or secondary, which is inapparent without protease activation 
of the packaged stocks of virus and subsequent infection of cells. This is because 
the rare revertant RNA generated during a packaging reaction is surrounded by 
a vast excess of nonrevertant genomes, and hence will be packaged with 
nonrevertant coat proteins. Thus the new phenotype is manifest only on 
subsequent infection.) 
[4521 
Recombinant DNA Research, Volume 18 
