122. Generation of a Family of Ad5 Mutants with Slowed Packaging Kinetics but Non-Affected Packaging Efficiency

Abstract

Adenoviral packaging is a multistep process where different viral proteins interact with the packaging signal Y to generate a DNA-protein complex which is driven to the procapsid to generate a viral particle. More than location of Y in the adenoviral (Ad) genome, the distance between the Inverted Terminal Repeat (ITR) and Y is crucial for the packaging process. Thus, while separations of 270 bp or less do not seem to affect packaging, distances of 600 bp or more significantly prevent efficient Ad genome packaging. To better understand the packaging process, we have developed a family of Ad mutant vectors with different combinations of short recombinase-recognized-sequences to flank Y. Sequences length's range from 67 to 109 bp, which theoretically should not affect the packaging process. However, in contrast to previous results, insertion of these sequences between 5-end ITR and Y lengthens viral cycle from 36 up to 52-56 h. Moreover, at 36 h, viral production was 2-3 logs lower in mutant Ad than in control, which may have important applications to separate defective from non-defective Ad. We then analyzed which steps of the packaging process were altered. Thus, Dot blot analysis through the viral cycle showed that DNA replication levels and kinetics were similar between control and mutant Ad. When cotransfecting with control and mutant Ad, total infection units (IU) of control Ad were again 2-3 logs higher than mutant Ad in packaging showing that protein production or protein availability are not a key factor to explain slower viral cycle. We then performed a packaging assay to compare viral replicated DNA vs packaged DNA and we observed that total replicated DNA levels between control and mutant Ad were similar but only 3-4% of the mutant genome was packaged compared to control, indicating a relevant impairment in the packaging process which could explain the significant delay of the viral cycle. In addittion, at the end of their viral cycle, total number of virus particles as well as IU/PP ratio were similar between control and mutant Ad demonstrating that packaging kinetics but not packaging efficiency was affected. Finally, ongoing experiments are under way to specifically determine which step of the interaction between packaging complex:Y is affected. Adenoviral packaging is a multistep process where different viral proteins interact with the packaging signal Y to generate a DNA-protein complex which is driven to the procapsid to generate a viral particle. More than location of Y in the adenoviral (Ad) genome, the distance between the Inverted Terminal Repeat (ITR) and Y is crucial for the packaging process. Thus, while separations of 270 bp or less do not seem to affect packaging, distances of 600 bp or more significantly prevent efficient Ad genome packaging. To better understand the packaging process, we have developed a family of Ad mutant vectors with different combinations of short recombinase-recognized-sequences to flank Y. Sequences length's range from 67 to 109 bp, which theoretically should not affect the packaging process. However, in contrast to previous results, insertion of these sequences between 5-end ITR and Y lengthens viral cycle from 36 up to 52-56 h. Moreover, at 36 h, viral production was 2-3 logs lower in mutant Ad than in control, which may have important applications to separate defective from non-defective Ad. We then analyzed which steps of the packaging process were altered. Thus, Dot blot analysis through the viral cycle showed that DNA replication levels and kinetics were similar between control and mutant Ad. When cotransfecting with control and mutant Ad, total infection units (IU) of control Ad were again 2-3 logs higher than mutant Ad in packaging showing that protein production or protein availability are not a key factor to explain slower viral cycle. We then performed a packaging assay to compare viral replicated DNA vs packaged DNA and we observed that total replicated DNA levels between control and mutant Ad were similar but only 3-4% of the mutant genome was packaged compared to control, indicating a relevant impairment in the packaging process which could explain the significant delay of the viral cycle. In addittion, at the end of their viral cycle, total number of virus particles as well as IU/PP ratio were similar between control and mutant Ad demonstrating that packaging kinetics but not packaging efficiency was affected. Finally, ongoing experiments are under way to specifically determine which step of the interaction between packaging complex:Y is affected.