HIV-1 Tropism Dynamics and Phylogenetic Analysis from Longitudinal Ultra-Deep Sequencing Data of CCR5- and CXCR4-Using Variants Mariano M. Sede 1,2 , Franco A. Moretti 1,2 , Natalia L. Laufer 1,2 , Leandro R. Jones 2,3. , Jorge F. Quarleri 1,2 * . 1 Instituto de Investigaciones Biome ´dicas en Retrovirus y Sida (INBIRS), Universidad de Buenos Aires, CONICET, Buenos Aires, Argentina, 2 Consejo de Investigaciones Cientı ´ficas y Te ´cnicas (CONICET), Buenos Aires, Argentina, 3 Laboratorio de Virologı ´a y Gene ´tica Molecular, Facultad de Ciencias Naturales, sede Trelew, Universidad Nacional de la Patagonia San Juan Bosco, Chubut, Argentina Abstract Objective: Coreceptor switch from CCR5 to CXCR4 is associated with HIV disease progression. The molecular and evolutionary mechanisms underlying the CCR5 to CXCR4 switch are the focus of intense recent research. We studied the HIV-1 tropism dynamics in relation to coreceptor usage, the nature of quasispecies from ultra deep sequencing (UDPS) data and their phylogenetic relationships. Methods: Here, we characterized C2-V3-C3 sequences of HIV obtained from 19 patients followed up for 54 to 114 months using UDPS, with further genotyping and phylogenetic analysis for coreceptor usage. HIV quasispecies diversity and variability as well as HIV plasma viral load were measured longitudinally and their relationship with the HIV coreceptor usage was analyzed. The longitudinal UDPS data were submitted to phylogenetic analysis and sampling times and coreceptor usage were mapped onto the trees obtained. Results: Although a temporal viral genetic structuring was evident, the persistence of several viral lineages evolving independently along the infection was statistically supported, indicating a complex scenario for the evolution of viral quasispecies. HIV X4-using variants were present in most of our patients, exhibiting a dissimilar inter- and intra-patient predominance as the component of quasispecies even on antiretroviral therapy. The viral populations from some of the patients studied displayed evidences of the evolution of X4 variants through fitness valleys, whereas for other patients the data favored a gradual mode of emergence. Conclusions: CXCR4 usage can emerge independently, in multiple lineages, along the course of HIV infection. The mode of emergence, i.e. gradual or through fitness valleys seems to depend on both virus and patient factors. Furthermore, our analyses suggest that, besides becoming dominant after population-level switches, minor proportions of X4 viruses might exist along the infection, perhaps even at early stages of it. The fate of these minor variants might depend on both viral and host factors. Citation: Sede MM, Moretti FA, Laufer NL, Jones LR, Quarleri JF (2014) HIV-1 Tropism Dynamics and Phylogenetic Analysis from Longitudinal Ultra-Deep Sequencing Data of CCR5- and CXCR4-Using Variants. PLoS ONE 9(7): e102857. doi:10.1371/journal.pone.0102857 Editor: Golo Ahlenstiel, University of Sydney, Australia Received December 16, 2013; Accepted June 25, 2014; Published July 17, 2014 Copyright: ß 2014 Sede et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study has been funded by National Agency of Scientific and Technological Promotion(ANPCYT) grant 0422 [http://www.agencia.mincyt.gob.ar/ frontend/agencia/convocatoria/105]. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * Email: quarleri@fmed.uba.ar . These authors contributed equally to this work. Introduction Human immunodeficiency virus type 1 (HIV-1) entry into host cells requires synchronized interactions of the envelope glycopro- tein gp120 with the CD4 receptor and with one of the chemokine receptors, CCR5 or CXCR4. HIV-1 tropism for the chemokine receptors CCR5 and CXCR4 has been shown to be associated with disease progression [1]. Viruses, especially those using the CCR5 receptor to enter the target cells (R5 viruses), are generally predominant at early stages of HIV-1 infection, whereas the emergence of CXCR4-using viruses (X4 viruses) generally occurs at later stages [2]. Variation in R5 Env proteins can also influence the ability of a virus to utilize various levels of CD4 and CCR5 found in different cell types, such as macrophages and T-cells. The presence of X4 viruses is consistently associated with low CD4+ T- cell counts and accelerated disease progression, although it is still unclear whether it is the cause or consequence of disease progression [3,4]. Inferring HIV-1 coreceptor usage from a genotype is becoming more and more important for appropriately treating long-term patients as the level of CXCR4-using viruses is associated with risk of virological failure to maraviroc-containing regimens in a dose-dependent fashion [5,6]. The primary genetic determinant for the HIV coreceptor usage is the third variable region (V3) of the HIV gp120 envelope glycoprotein encompassing approximately 35 residues (varying in length depending on the viral isolate) with a conserved disulfide PLOS ONE | www.plosone.org 1 July 2014 | Volume 9 | Issue 7 | e102857