Human immunodeficiency virus type 1 cloning vectors for antiretroviral resistance testing

Javier Martinez-Picado, Lorraine Sutton, Maria Pia De Pasquale, Anu V. Savara, Richard T. D'Aquila*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

35 Scopus citations

Abstract

Better detection of minority human immunodeficiency virus type 1 (HIV-1) populations containing gene mutations may improve the usefulness of antiretroviral resistance testing for clinical management. Molecular cloning of HIV-1 PCR products which might improve minority detection can be slow and difficult, and commercially available recombinant virus assays test drug susceptibility of virus pools. We describe novel plasmids and simple methods for rapid cloning of HIV-1 PCR products from patient specimens and their application to generate infectious recombinant virus clones for virus phenotyping and genotyping. Eight plasmids with differing deletions of sequences encoding HIV-1 protease, reverse transcriptase, or Gag p7/p1 and Gag p1/p6 cleavage sites were constructed for cloning HIV-1 PCR products. A simple HIV-1 sequence-specific uracil deglycosylase-mediated cloning method with the vectors and primers designed here was more rapid than standard ligase-mediated cloning. Pooled and molecularly cloned infectious recombinant viruses were generated with these vectors. Replicative viral fitness and drug susceptibility phenotypes of cloned infectious viruses containing patient specimen-derived sequences were measured. Clonal resistance genotyping analyses were also performed from virus isolates, plasma HIV-1 RNA, and infected cell DNA. Sequencing of a limited number of molecular clones detected minorities of resistant virus not identified in the pooled population PCR product sequence and linkage of minority mutations.

Original languageEnglish (US)
Pages (from-to)2943-2951
Number of pages9
JournalJournal of clinical microbiology
Volume37
Issue number9
DOIs
StatePublished - 1999

ASJC Scopus subject areas

  • Microbiology (medical)

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