In-depth analysis of G-to-A hypermutation rate in HIV-1 env DNA induced by endogenous APOBEC3 proteins using massively parallel sequencing

• Published in: Journal of virological methods Vol. 171; no. 2; pp. 329 - 338
• Main Authors: Knoepfel, Stefanie A., Di Giallonardo, Francesca, Däumer, Martin, Thielen, Alexander, Metzner, Karin J.
• Format: Journal Article
• Language: English
• Published: Kidlington Elsevier B.V 01.02.2011; Elsevier
• Subjects: adenine; APOBEC Deaminases; APOBEC3; Biological and medical sciences; Cells, Cultured; Cytidine Deaminase; Cytosine Deaminase - immunology; Cytosine Deaminase - metabolism; deamination; DNA; DNA Mutational Analysis; DNA, Viral - genetics; env Gene Products, Human Immunodeficiency Virus - genetics; Fundamental and applied biological sciences. Psychology; G-to-A hypermutation; genome; High-Throughput Nucleotide Sequencing; HIV-1; HIV-1 - genetics; HIV-1 - growth & development; HIV-1 envelope; Human immunodeficiency virus 1; Humans; Leukocytes, Mononuclear - virology; Massively parallel sequencing; Microbiology; mutation; Point Mutation; Primary CD4+ cells; proteins; Sequence Analysis, DNA; T-lymphocytes; T-Lymphocytes - virology; Techniques used in virology; Virology; HIV-1; Massively parallel sequencing; APOBEC3; HIV-1 envelope; G-to-A hypermutation; Primary CD4+ cells; HIV-1 virus; Retroviridae; Method; Lentivirus; Protein; Primary CD4; Virus; Endogenous; cells; Human immunodeficiency virus
• ISSN: 0166-0934; 1879-0984
• DOI: 10.1016/j.jviromet.2010.11.016

Some APOBEC3 proteins cause G-to-A hypermutation in HIV-1 DNA when the accessory viral protein Vif is absent or non-functional. So far, cloning and sequencing has been performed to study G-to-A hypermutation. This is time-consuming and labour-intensive especially in the context of in vivo investigations where the number of hypermutated sequences can be very low. Thus, a massively parallel sequencing protocol has been developed for in-depth analysis of G-to-A hypermutation using the 454 pyrosequencing FLX system. Part of HIV-1 env was amplified and pyrosequenced after two rounds of infection in T cell lines and PBMCs using HIV-1 NL4-3Δvif. Specific criteria were applied to cope with major technical challenges: (1) the inclusion of hypermutated sequences, (2) the high genome diversity of HIV-1 env, and (3) the exclusion of sequences containing frameshift errors caused by pyrosequencing. In total, more than 140,000 sequences were obtained. 1.3–6.5% of guanines were mutated to adenine, most frequently in the GG dinucleotide context, the preferred deamination site of APOBEC3G. Non-G-to-A mutations occurred only in low frequencies (<0.6%). Single hypermutated sequences contained up to 24 G-to-A mutations. Overall, massively parallel sequencing is a very useful tool for in-depth analysis of G-to-A hypermutation in HIV-1 DNA induced by APOBEC3 proteins.