Human immunodeficiency virus type 1 cloning vectors for antiretroviral resistance testing

Better detection of minority human immunodeficiency virus type 1 (HIV-1) po pulations containing gene mutations may improve the usefulness of antiretro viral resistance testing for clinical management. Molecular cloning of HIV- 1 PCR products which might improve minority detection can be slow and diffi cult, and commercially available recombinant virus assays test drug suscept ibility of virus pools. We describe novel plasmids and simple methods for r apid cloning of HIV-1 PCR products from patient specimens and their applica tion to generate infectious recombinant virus clones for virus phenotyping and genotyping, Eight plasmids with differing deletions of sequences encodi ng HIV-1 protease, reverse transcriptase, or Gag p7/p1 and Gag p1/p6 cleava ge sites were constructed for cloning HIV-1 PCR products. A simple HIV-1 se quence-specific uracil deglycosylase-mediated cloning method with the vecto rs and primers designed here was more rapid than standard ligase-mediated c loning. Pooled and molecularly cloned infectious recombinant viruses were g enerated with these vectors. Replicative viral fitness and drug susceptibil ity phenotypes of cloned infectious viruses containing patient specimen-der ived sequences were measured. Clonal resistance genotyping analyses were al so performed from virus isolates, plasma HIV-1 RNA, and infected cell DNA, Sequencing of a limited number of molecular clones detected minorities of r esistant virus not identified in the pooled population PCR product sequence and linkage of minority mutations.