Recovery of Virtually Full-Length HIV-1 Provirus of Diverse Subtypes from Primary Virus Cultures Using the Polymerase Chain Reaction

Abstract

In the course of the global pandemic, the human immunodeficiency virus type-1 (HIV-1) has established at least eight distinct genotypes in the main (M), or prevalent, group of isolates, a variety of rare outlier forms, and intergenotypic recombinants of group M viruses. This genotypic diversity has been documented, for the most part, by sequencing of subgenomic segments of the provirus. Using DNA from virus cultures on peripheral blood mononuclear cells (PBMC) and recent improvements of the PCR technique, we have amplified virtually full-length HIV-1 genomes from genetic subtypes A through G of group M viruses and molecularly cloned several of them. Resequencing of the complete genome of a prototype strain after long PCR amplification and cloning has established a PCR error rate of 0.14%. We also report the first complete PCR-derived sequence of a U.S. clinical isolate of genotype B expanded only in primary PBMC; this provirus harbors a uniquely truncated V3 loop.

Transition between Stochastic Evolution and Deterministic Evolution in the Presence of Selection: General Theory and Application to Virology

“…Compensatory mutations, which become advantageous only after initial resistance mutations occur, have also observed in HIV-infected patients treated with protease inhibitors (11). The development of molecular techniques that allow full-length HIV genomes to be sequenced (69) means that it is only a matter of time before genetic data become available to study the evolutionary processes of linkage disequilibrium, background selection, and compensation in infected individuals.…”

Virus 'quasispecies': making a mountain out of a molehill?

“…For example, amongst 28 clones of the human HLA-DPβ gene (239 nt), 17 introduced substitutions were observed (Saiki et al, 1988), giving an error rate after 30 cycles of PCR of 0n85i10 −% \nt\cycle. Another estimate is provided by comparison of full-length genome copies of HIV-1 generated by 25 cycles of PCR in which 13\9000 positions differed because of PCR errors, equivalent to an error rate of 0n6i10 −% (Salminen et al, 1995). We have also estimated the error rate of Taq by amplifying cDNA for the HCV core gene at a dilution where only 1 out of 10 replicate PCR reactions was positive.…”

Biologic and Genetic Characterization of a Panel of 60 Human Immunodeficiency Virus Type 1 Isolates, Representing Clades A, B, C, D, CRF01_AE, and CRF02_AG, for the Development and Assessment of Candidate Vaccines

“…PCR amplification, complete genome sequencing, and envelope sequencing of HIV-1 strains was performed as previously described (43,59,61). The GenBank accession numbers for the 60 (21 new) complete genomes and for the 41 new envelope sequences from the virus cultures reported here are all listed (see Fig.…”