Computational protocol: Relaxation of Adaptive Evolution during the HIV-1 Infection Owing to Reduction of CD4+ T Cell Counts

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Protocol publication

[…] The sequences were initially aligned using the ClustalX program , then sites with deletions and insertions were then excluded in order to preserve the reading frames of the genes. After this editing process, the sequences were manually aligned using the SE-AL program, version 2.0 (Department of Zoology, Oxford University; To compute pairwise distances we used the Kimura two-parameters model, the estimates were performed using the Mega software version 4.0 [...] To construct the trees we used Bayesian methods, assuming GTR and a gamma correction model, as is implemented in the MrBayes program, version 3.1.2 . Two independent Markov chain Monte Carlo (MCMC) processes were run for 2×107 generations with the initial 10% of each run discarded as burn-in. The convergence of chains was evaluated using the TRACER software, version 1.2 (, and runs were accepted when all parameters presented the effective sample size number (ESS) greater than 100. Maximum posteriori (MP) trees were show and edited using the FigTree software ( [...] Selective pressures were analyzed using an approach that estimates the number of dN and dS at all sites in the sequence alignments. The method compares the fit to the data of various models of codon evolution, which differ in the distribution of dN/dS (ω) among codons and takes into account the phylogenetic relationships of the sequences .The model 0 (M0: one-ratio) assumes a single ω for all codons in the alignment and hence is the simplest model specified.The model 1 (M1: neutral) allows different proportions of conserved (ω = 0) and neutral codons (ω = 1), both estimated from the data. The model 2 (M2: selection) has an additional class of codons with its ω ratio (which can be >1) estimated from the data. The model 3 (M3: discrete) also allows positive selection by incorporating three categories of codons with the ω value at each estimated from the data. Nested models can be compared using a standard likelihood ratio test (LRT). Significant evidence for positive selection is provided if M2, or more normally M3, significantly reject the null hypothesis M0 and M1, and if the favored models contain a class of codons where ω>1. We also used an extension of M3 to investigate if there are sites evolving under different selective pressure, this model allow to detected sites not necessarily under positive selection . This model (model D with k = 3) was used to determine if a specific group of sequences (i.e., sequences of recipient RB sampled in 1990) was subjected to a distinct selective regimen in a phylogenetic tree constructed with sequences sampled over the HIV-1 infection time in individual. Since model D is nested to M3, we used LRT and M3 as the null hypothesis assuming one degree of freedom. All these models are implemented in the CODEML program from the PAML v. 3.14 package ( To the positive selection estimate molecular clones of each patient were analyzed separately. Additional subdivisions of the intra-host molecular clones were also made in order to obtain more detailed information on a particular time interval. For example, sequences of the donor DO were first analyzed in a time interval between 1985 and 1993. Then these sequences were subdivided into two other datasets; one composed by HIV-1 variants with the GPGR tetramer at the V3 loop of env gene and other composed by variants with GSGR tetramer. These two datasets included clones sampled between 1987 and 1991 because GSGR viruses emerged after 1987 (see results). […]

Pipeline specifications

Software tools Clustal W, Se-Al, MEGA, MrBayes, BEAST, FigTree, PAML
Applications Phylogenetics, Nucleotide sequence alignment
Organisms Human immunodeficiency virus 1, Human immunodeficiency virus 2, Homo sapiens
Diseases Acquired Immunodeficiency Syndrome, Infection, HIV Infections