Depending on when during the packaging reaction the recombination event occurs, each resulting 
replication-competent virus could amplify itself in the same cell or adjacent cells by as much as 
a hundred fold, or more. Thus the observed frequency of recombinants is a maximum value: 
the actual rate of recombination is likely much lower. This mechanism also explains the wide 
variability observed in frequency of recombinants among the different virus stocks tested. 
3. CLEAVAGE-DEFICIENT VARIANTS OF THE E2 SPIKE PROTEIN 
The evidence for low levels of replication-competent virus in cells infected with 
SFV3/Helperl virion suggested the value of additional measures to enhance the 
biosafety of this gene expression system. Liljestrom and colleagues have shown 
that conditionally infectious particles (dependent on protease activation for 
infectivity) can be produced using a cleavage-deficient variant of SFV (Salminen, 
et al., 1992*). During transport of the p62-El spike heterodimer from the 
endoplasmic reticulum to the cell surface, p62 is cleaved to E2 and E3 in a post- 
Golgi compartment. Cleavage of p62 can be abolished by a single Arg for Leu 
substitution (mL mutation) in the last amino acid residue position of the E3 
moiety of the p62 protein (Lobigs and Garoff, 1990; Salminen, et al., 1992*) 
(Figure 7). The mL mutation does not significantly affect virus maturation and 
budding, but the virus produced is virtually noninfectious, due to reduced binding 
and uptake into endosomes, as well as to reduced frequency of fusion between the 
viral and endosomal membranes. To infect cells with packaged virions, the virus 
stock is activated using chymotrypsin, which probably cleaves p62 at the leucine 
residue of the mutated cleavage site. 
In separate, whole-animal studies (Glasgow et al., 1991*), the mL-SVF variant 
demonstrated a markedly reduced virulence for 40-day-old Balb/c mice when 
compared to the parent, unmutated cDNA clone (SFV4) or to the attenuated 
substrain of L10, from which the original cDNA clone was derived (Table 3). 
These results demonstrate that mutations which block p62 cleavage effectively 
reduce the virulence of SFV. 
To reduce the potential for genetic reversion to wild-type infectivity, another 
mutant, SFV-SQL (Figure 7), was constructed in which all the arginine residues 
at the cleavage site were changed. Serine and glutamine substitutions were 
chosen, since such substitutions have been shown not to affect virus assembly 
(Jain et al., 1991). In addition, to reduce the possibility of reversion, two of the 
mutations were based on three-base substitutions and one on a two-base 
substitution. 
Recombinant DNA Research, Volume 18 
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