Background During HIV-1 infection coreceptor switch from CCR5- (R5)- to CXCR4 (X4)-using viruses is associated with disease progression. thymus. Conclusions/Significance Our data indicate that the evolution of X4 strains is a multi-step, temporally structured process and that the thymus may play an important role in the evolution/amplification of coreceptor variants. Development of new therapeutic protocols targeting virus in the thymus could be important to control HIV-1 infection prior to advanced disease. Introduction Infection of target cells by Ansamitocin P-3 human Ansamitocin P-3 immunodeficiency virus type 1 (HIV-1) requires binding of Ansamitocin P-3 the viral surface protein gp120 to the cellular surface protein CD4 and chemokine receptors CCR5 or CXCR4 [1]. R5 viruses using the CCR5 coreceptor represent the predominant viral quasispecies during the early and chronic phases of the infection [2], [3]. X4 viruses using the CXCR4 coreceptor appear at a later stage in about 50% of individuals infected by HIV-1 subtype B and are associated with accelerated MGC3199 disease progression [4], [5]. The reasons for coreceptor evolution during the course of infection and the origin and evolution of X4 strains are not fully understood, although several hypotheses have been proposed [6]. Appearance of X4 viruses might reflect emergence of quasispecies sequestered in tissues at the time of infection [7] or evolution from R5 viruses [8]C[10]. The primary genetic determinants of HIV-1 coreceptor use are concentrated within the 35-amino acid hypervariable V3 loop of the envelope protein gp120 [11]C[13]. Although a small number of basic amino acid substitutions in V3 may be sufficient for changes in coreceptor preference, combinations of V3 mutations can lead to major loss of entry fitness in culture, unless compensated by mutations in or near V1-V2 in gp120 [14], indicating that complex, discontinuous determinants contribute to X4 coreceptor use, at least on certain cell types [9], [15], [16]. Continuing HIV-1 replication in anatomic or cellular reservoirs and release of latent virus from infected reservoirs can contribute to viral rebound following interruption of combination anti-retroviral therapy (ART) [17], [18]. Genital tissues and blood appear to serve as distinct reservoirs harboring latent HIV-1 during prolonged drug therapy [19], [20], while the brain is a viral compartment harboring HIV-1 subpopulations with specific genetic characteristics [21]C[26]. CD4 T lymphocytes in infants and children predominantly express CD45RA, whereas in adults about equal ratios of CD45RA or CD45RO are expressed [27]. Only a subset of activated CD4 CD45RO T cells express CCR5, while the preponderance of CD4 T-lymphocytes, independent of CD45 isoform, express CXCR4 coreceptors [27], [28]. The thymus harbors a large number of immature and mature CD4 thymocytes expressing CXCR4, but relatively limited CCR5-expressing cells, implicating the thymus as a critical compartment for HIV-1 pathogenesis [29]C[32]. X4 viral strains are highly cytopathic to immature thymocytes [33]. Within HIV-1 infected individuals, significant reduction in thymocyte proliferation, output and function occurs in the absence of ART [34], [35], while HIV-induced destruction of the thymus decreases the capacity for T-cell immune reconstitution resulting in rapid disease progression in infected children [36]. Despite the importance of X4 strains for pathogenesis, virtually no studies have evaluated coreceptor use or the evolutionary patterns across hypervariable regions of HIV-1 quasispecies infecting the thymus [37]. Recently, a to analyze HIV-1 subpopulations (tissues and PBMCs of different subjects. Analysis of recombinant sequences HIV-1 frequently recombines [42]C[44]. Since intra-patient recombination would lead to the creation of mosaic genomes violating the tree-like assumption of evolution, we carefully.