Whole-genome characterization of human and simian immunodeficiency virus intrahost diversity by ultradeep pyrosequencing

Abstract

Rapid evolution and high intrahost sequence diversity are hallmarks of human and simian immunodeficiency virus (HIV/SIV) infection. Minor viral variants have important implications for drug resistance, receptor tropism, and immune evasion. Here, we used ultradeep pyrosequencing to sequence complete HIV/SIV genomes, detecting variants present at a frequency as low as 1%. This approach provides a more complete characterization of the viral population than is possible with conventional methods, revealing low-level drug resistance and detecting previously hidden changes in the viral population. While this work applies pyrosequencing to immunodeficiency viruses, this approach could be applied to virtually any viral pathogen.

FIG. 1.

Genome-wide pyrosequencing of SIV. (A) Schematic representation of SIV amplification strategy and fragmentation. (B) Sequence coverage obtained for each SIV genome. Gray bars above graph denote RT-PCR amplicons. Sequence position is based on the SIVmac239 consensus reference sequence. (C) Graphs indicate the percentage of nucleotides that are non-wild-type at each position at 54 (top) and 82 (bottom) weeks postinfection. Asterisks above the graph for 54 wpi indicate mutations detected by conventional Sanger sequencing at that time point. The dotted lines represent mutations identified at a frequency of less than 10%. (D) Changes in mutation frequency between 54 and 82 weeks postinfection. (E) Graph of mutation frequency for each position of an SIVmac239 stock. (F) Summary of nonsynonymous and synonymous mutation frequencies from 54- and 82-wpi genomes. A mutation summary is also shown for the SIVmac239 stock. NT, nucleotide.

FIG. 2.

Cytotoxic T-lymphocyte escape. (A) Sequences of epitopes restricted by Mamu-A1*001 and Mamu-A1*002 with high levels of mutation. An SIVmac239 stock is included as a control. Mutations are color coded by frequency. (B) Sequences of epitopes restricted by Mamu-A1*001 and Mamu-A1*002 with low levels of mutation.

FIG. 3.

Genome-wide pyrosequencing of HIV. Sequence coverage obtained across the genome of a representative set of four HIV genomes and an HIV plasmid is shown. Gray bars represent RT-PCR amplicons used to amplify the HIV coding region for pyrosequencing. Sequence position is based on the HXB2 consensus reference sequence. BR1005, BR1009, etc., are numbers used to identify patients in the study.

FIG. 4.

Variation within the Env V3 loop. Consensus sequences representing the major variants spanning the envelope V3 loop in patient 114 (A) and patient 115 (B) are shown. Nonsynonymous mutations are in yellow. Synonymous mutations are in green.