Construction of an infectious genomic clone of parvovirus LuIII with a deletion of the A/T rich sequence

Parvoviruses are small, single stranded DNA viruses. These viruses are currently being studied to be used as transient vectors in gene therapy. The encapsidation pattern of the autonomous parvovirus LuIII and others parvovirus is not completely understood. LuIII encapsidates plus and minus DNA strands with equal frequency. This parvovirus shares over 80% sequence identity with Mice Minute Virus (MMv) and H-1, rodent parvoviruses, that encapsidate primarily 99% minus strand DNA. A unique A/T rich region of 47 nucleotides at map unit 89, only exists in the LuIII genome. The location of this unique A/T sequence and its absence from genomes of closely related parvoviruses, suggest that this element could be a putative encapsidation signal.;To study the role of this unique sequence a mutated genomic clone of LUIII (pGLU A/T-) was constructed by deleting the A/T rich sequence from the clone pGLU883 (pUC 19 nucleotide (nt): 2686, LuIII nt: 1--5135) by using the polymerase chain reaction method (PCR) and conventional DNA recombinant techniques. A fragment of 590 base pairs (bp) (pUC19 nt: 352--416; LuIII nt: 4609--5135) and a fragment of 4631bp (pUC19 nt: 416--486; LuIII nt: 1--4561) were obtained by PCR amplification. The 590bp fragment had a KpnI site at nt 408 and a ClaI site at nt 4609 and the 4631 bp fragment has a SalI site at nt 429 and a ClaI site at nt 4561. The fragments were subjected to restriction enzyme digestion with KpnI and SalI and ligated to pUC19 previously digested with KpnI and SalI After ligation the ClaI sites were cut and ligated to obtain closed molecules (clone pGLU A/T-). E. coli DH5alpha cells were transformed by electroporation.