[Generation of a series of recombinant herpes simplex viruses which can provide replicating and packaging functions for recombinant adeno-associated virus].
BACKGROUND
To construct a series of recombinant herpes simplex viruses that can provide replicating and packaging functions for recombinant adeno-associated virus (rAAV), and to select the strains possessing stronger functions for large-scale production of rAAV.


METHODS
A set of cosmids that represents the whole genome of HSV-1 was used to generate recombinant HSV-1 expressing rep and cap proteins of AAV-2. An ATG-to-ACG mutation in the start codon of AAV-2 rep protein was generated by site-directed mutagenesis. rep and cap genes, under control of their native promoters, with or without the ATG-to-ACG mutation in the start codon of rep, were inserted into the Xba   site of UL2 or UL44 genes, respectively, resulting in the recombinant cosmids cos6-rmc/  UL2, cos56-rc/  UL44 and cos56-rmc/  UL44. Homologous recombination among the HSV-1 fragment on the recombinant cosmids and the rest fragments of HSV-1 genome resulted in three strains of recombinant HSV-1. Together with the one was constructed previously, there were four strains of recombinant HSV-1named HSV1-rc/  UL2, HSV1-rmc/  UL2, HSV1-rc/  UL44 and HSV1-rmc/  UL44 respectively.


RESULTS
PCR detection confirmed the existence of   rep- gene in the genomes of all four strains of the recombinant HSV-1. Recombinant AAV was produced after infecting the AAV vector cell line that carrying the GFP expression cassette with the four strains of recombinant HSV-1 respectively. However, HSV1-rc/  UL2 and HSV1-rmc/  UL2 produced much more rAAV than HSV1-rc/  UL44 and HSV1c/  UL44 did.


CONCLUSIONS
All the four strains of recombinant HSV-1 support rAAV replication and packaging. HSV1  UL2 and HSV1-rmc/  UL2 that provide much stronger functions may be useful for large-scale production of rAAV.
Wu
Zhijian
Z
State Laboratory of Molecular Virology and Genetic Engineering, Institute of Virology, Chinese Academy of Preventive Medicine, Beijing 100052, China.
Wu
Xiaobing
X
Hou
Yunde
Y
D000818:Animals; D002478:Cells, Cultured; D003360:Cosmids / Q000235:genetics; D006224:Cricetinae; D000229:Dependovirus / Q000235:genetics* / Q000502:physiology; D005822:Genetic Vectors; D018259:Herpesvirus 1, Human / Q000235:genetics* / Q000502:physiology; D011995:Recombination, Genetic; D014162:Transfection; D014779:Virus Replication