Charge-to-Alanine Mutagenesis of the Adeno-Associated Virus Type 2 Rep78/68 Proteins Yields Temperature-Sensitive and Magnesium-Dependent Variants

Abstract

The adeno-associated virus type 2 (AAV) replication (Rep) proteins Rep78 and 68 (Rep78/68) exhibit a number of biochemical activities required for AAV replication, including specific binding to a 22-bp region of the terminal repeat, site-specific endonuclease activity, and helicase activity. Individual and clusters of charged amino acids were converted to alanines in an effort to generate a collection of conditionally defective Rep78/68 proteins. Rep78 variants were expressed in human 293 cells and analyzed for their ability to mediate replication of recombinant AAV vectors at various temperatures. The biochemical activities of Rep variants were further characterized in vitro by using Rep68 His-tagged proteins purified from bacteria. The results of these analyses identified a temperature-sensitive (ts) Rep protein (D40,42,44A-78) that exhibited a delayed replication phenotype at 32°C, which exceeded wild-type activity by 48 h. Replication activity was reduced by more than threefold at 37°C and was undetectable at 39°C. Stability of the Rep78 protein paralleled replication levels at each temperature, further supporting ats phenotype. Replication differences resulted in a 3-log-unit difference in virus yields between the permissive and nonpermissive temperatures (2.2 × 106 and 3 × 103, respectively), demonstrating that this is a relatively tight mutant. In addition to the ts Rep mutant, we identified a nonconditional mutant with a reduced ability to support viral replication in vivo. Additional characterization of this mutant demonstrated an Mg2+-dependent phenotype that was specific to Rep endonuclease activity and did not affect helicase activity. The two mutants described here are unique, in that Rep tsmutants have not previously been described and the D412A Rep mutant represents the first mutant in which the helicase and endonuclease functions can be distinguished biochemically. Further understanding of these mutants should facilitate our understanding of AAV replication and integration, as well as provide novel strategies for production of viral vectors.

Mutagenesis of the dengue virus type 2 NS3 proteinase and the production of growth-restricted virus

“…The remaining three viruses grew to reach good titres (Fig. 4) and displayed small plaques but did not show the temperature-sensitive phenotype that has been observed for some viruses with charged-to-alanine mutations in non-structural genes (Diamond & Kirkegaard, 1994 ; Parkin et al , 1996 ; Muylaert et al , 1997 ; Huang et al , 1998 ; Gavin et al , 1999 ). Virus V 32–36 replicated too poorly to be of further use and therefore the viruses of most interest with respect to growth restriction were V 63–66 and V 91–94 .…”

“…Fewer mutants have been reported for other viruses following charged-to-alanine mutagenesis. Single temperature-sensitive mutants have been described for Human immunodeficiency virus with mutations located in the reverse transcriptase and integrase genes (Wiskerchen & Muesing, 1995 ; Huang et al , 1998 ), and Adeno-associated virus type 2 following mutagenesis of the Rep78/68 helicase genes (Gavin et al , 1999 ). Mutagenesis of the polymerase subunit PB2 of Influenza A virus generated three temperature-sensitive mutants after mutagenesis of ten sites (Parkin et al , 1996 ).…”

Comprehensive mutagenesis maps the effect of all single codon mutations in the AAV2repgene on AAV production

“…The sequence of the rep genes in naturally occurring serotypes is relatively well conserved (average of 78% amino acid sequence identity between AAV2 rep and AAV1-13 rep ). While previous mutational studies of the Rep proteins have been conducted, these studies have focused primarily on identifying non-functional Rep variants to elucidate the location of key motifs (Davis et al, 2000; Gavin et al, 1999; Hörer et al, 1995; Yang et al, 1992). As such, Rep engineering efforts may benefit from the exploration of additional sequence diversity.…”