Computational protocol: Functional conservation and coherence of HIV-1 subtype A Vpu alleles

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Protocol publication

[…] Blood samples were collected from 10 HIV-1 positive individuals described in Table . Viral RNA was extracted from the plasma samples using the QIAamp UltraSens Virus Kit (Qiagen) according to the manufacturer’s instructions and stored at −80 °C. Viral RNA was reverse transcribed into cDNA using HIV-1-env-R (5′-CTT CCT GCT GCT CCT AAG AAC CCA-3′) with SuperScript II Reverse Transcriptase (ThermoFisher Scientific). To amplify the vpu gene, cDNA was then amplified using HIV-1-tat-F (5′-ATT TCC TAT GGC AGG AAG AAG CGG A-3′) and HIV-1-env-R with the Promega GoTaq Flexi Kit (Promega). The primers amplified a fragment which was about 1800 nucleotides in length. To amplify the pol gene, cDNA was amplified using HIV-1-pol-F (AGG ACA CCA AAT GAA AGA CTG CAC T) and HIV-1-pol-R (CAT ATT TCT TCC AAT TAT GTT GAC A). The primers amplified a fragment which was about 460 nucleotides in length. This fragment was chosen such that it contained the previously described recombination site of CRF35_AD. PCR products were sequenced using the BigDye Terminator v3.1 Cycle Sequencing Kit (ThermoFisher Scientific) and analyzed on an ABI Prism 3130 automated DNA sequencer (Applied Biosystems). Vpu (246 nucleotides) and the recombinant fragment of Pol (268 nucleotides) were trimmed and the nucleotide sequences were submitted to GenBank under accession numbers indicated in Table . Sequences were aligned with HIV-1 reference sequences obtained from LANL HIV Database, using Clustal X2. The alignments were manually verified using BioEdit version 7.2.5. MEGA version 7 was used to construct a neighbor-joining phylogenetic tree with a uniform rate of variation among sites and nucleotide substitution using the Kimura two-parameter. The reliability of the branching and clustering pattern was estimated from 1000 bootstrap replicates. The tree was rooted with the reference sequences of HIV-1 group N and SIVcpz. [...] To show the characteristics of the clinical isolates, consensus sequence of the subtype A Vpu alleles was obtained using WebLogo (http://weblogo.threeplusone.com/). […]

Pipeline specifications

Software tools Clustal W, BioEdit, MEGA, WebLogo
Applications Phylogenetics, Genome data visualization
Organisms Homo sapiens, Human poliovirus 1 Mahoney
Diseases HIV Infections