In-depth analysis of G-to-A hypermutation rate in HIV-1 env DNA induced by endogenous APOBEC3 proteins using massively parallel sequencing.
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.
Institute of Clinical and Molecular Virology, University of Erlangen-Nuremberg, Germany. S.A.Knoepfel@amc.uva.nl
Stefanie A
SA
Knoepfel
Francesca
F
Di Giallonardo
Martin
M
Däumer
Alexander
A
Thielen
Karin J
KJ
Metzner
D000071478:APOBEC Deaminases; D002478:Cells, Cultured; D003564:Cytidine Deaminase; D043525:Cytosine Deaminase / Q000276:immunology* / Q000378:metabolism; D004252:DNA Mutational Analysis; D004279:DNA, Viral / Q000235:genetics*; D015497:HIV-1 / Q000235:genetics* / Q000254:growth & development*; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000821:virology; D017354:Point Mutation; D017422:Sequence Analysis, DNA; D013601:T-Lymphocytes / Q000821:virology; D054299:env Gene Products, Human Immunodeficiency Virus / Q000235:genetics*
D004279:DNA, Viral; D054299:env Gene Products, Human Immunodeficiency Virus; D043525:Cytosine Deaminase; D000071478:APOBEC Deaminases; C502889:APOBEC3 proteins, human; D003564:Cytidine Deaminase