Monotypic human immunodeficiency virus type 1 genotypes across the uterine cervix and in blood suggest proliferation of cells with provirus

Abstract

Understanding the dynamics and spread of human immunodeficiency virus type 1 (HIV-1) within the body, including within the female genital tract with its central role in heterosexual and peripartum transmission, has important implications for treatment and vaccine development. To study HIV-1 populations within tissues, we compared viruses from across the cervix to those in peripheral blood mononuclear cells (PBMC) during effective and failing antiretroviral therapy (ART) and in patients not receiving ART. Single-genome sequences of the C2-V5 region of HIV-1 env were derived from PBMC and three cervical biopsies per subject. Maximum-likelihood phylogenies were evaluated for differences in genetic diversity and compartmentalization within and between cervical biopsies and PBMC. All subjects had one or more clades with genetically identical HIV-1 env sequences derived from single-genome sequencing. These sequences were from noncontiguous cervical biopsies or from the cervix and circulating PBMC in seven of eight subjects. Compartmentalization of virus between genital tract and blood was observed by statistical methods and tree topologies in six of eight subjects, and potential genital lineages were observed in two of eight subjects. The detection of monotypic sequences across the cervix and blood, especially during effective ART, suggests that cells with provirus undergo clonal expansion. Compartmentalization of viruses within the cervix appears in part due to viruses homing to and/or expanding within the cervix and is rarely due to unique viruses evolving within the genital tract. Further studies are warranted to investigate mechanisms producing monotypic viruses across tissues and, importantly, to determine whether the proliferation of cells with provirus sustain HIV-1 persistence in spite of effective ART.

FIG. 1.

Phylogenetic analysis of HIV-1 env sequences from all eight participants. Maximum-likelihood phylogenetic analyses of the HIV-1 env C2-V5 region derived by SGS of PBMC (open circles) and cervical DNA (rectangles) are shown for each subject. The cervical sequences are from three separate biopsies per subject. The biopsies correspond to positions on a clock with the os at the center: 3 o'clock (gray rectangles), 6 o'clock (black rectangles), and 9 o'clock (white rectangles). Numerals at nodes indicate bootstrap support for phylogenetic relationships. Subjects 1 to 3 were studied during effective (<50 copies/ml) ART, subjects 4 and 5 were studied during failing ART with active viral replication (>400 copies/ml), and subjects 6 to 8 were not receiving ART. Monotypic (identical) HIV-1 env sequences were detected in all subjects from noncontiguous regions of the uterine cervix and in the blood of subjects 1, 2, 5, 6, and 7, suggesting clonal expansion of HIV-1-infected cells. Examination of multiple biopsies compared to a single biopsy from each subject found increased compartmentalization (Table 3). Despite statistical evidence of compartmentalization, sequences within most clades included genital tract and blood viruses, suggesting genetic flow between the two tissues. Clades comprised of only genital sequences were observed in all participants who showed evidence of compartmentalized HIV-1 by one or more statistical test (subjects 1, 2, 4, 6, 7, and 8), regardless of whether a subject was receiving ART. Potential genital lineages, defined by a clade of diverse variants that are distant (>4%) from the nearest PBMC viruses, are framed in black boxes. Sequences were rooted using representative sequences for the corresponding subtype from the GenBank database (for clade B, sequences B.US.83.RF, B.US.90.WEAU160, B.FR.83.HXB2, B.US.86.JRFL; for clades A-E, sequences A1.KE.93.Q23-17, A1.SE.94.SE7253, A1.UG.92.92UG037, A1.UG.85.U455; and for clade D, sequences D.CD.83.ELI.K03454, D.CM.01.01CM4412HAL.AY371157, D.UG.94.94UG114.U88824, and D.TZ.01.01TZA280.AY253311). The scale bar (horizontal line) indicates the number of substitutions per site.

FIG. 1.

Phylogenetic analysis of HIV-1 env sequences from all eight participants. Maximum-likelihood phylogenetic analyses of the HIV-1 env C2-V5 region derived by SGS of PBMC (open circles) and cervical DNA (rectangles) are shown for each subject. The cervical sequences are from three separate biopsies per subject. The biopsies correspond to positions on a clock with the os at the center: 3 o'clock (gray rectangles), 6 o'clock (black rectangles), and 9 o'clock (white rectangles). Numerals at nodes indicate bootstrap support for phylogenetic relationships. Subjects 1 to 3 were studied during effective (<50 copies/ml) ART, subjects 4 and 5 were studied during failing ART with active viral replication (>400 copies/ml), and subjects 6 to 8 were not receiving ART. Monotypic (identical) HIV-1 env sequences were detected in all subjects from noncontiguous regions of the uterine cervix and in the blood of subjects 1, 2, 5, 6, and 7, suggesting clonal expansion of HIV-1-infected cells. Examination of multiple biopsies compared to a single biopsy from each subject found increased compartmentalization (Table 3). Despite statistical evidence of compartmentalization, sequences within most clades included genital tract and blood viruses, suggesting genetic flow between the two tissues. Clades comprised of only genital sequences were observed in all participants who showed evidence of compartmentalized HIV-1 by one or more statistical test (subjects 1, 2, 4, 6, 7, and 8), regardless of whether a subject was receiving ART. Potential genital lineages, defined by a clade of diverse variants that are distant (>4%) from the nearest PBMC viruses, are framed in black boxes. Sequences were rooted using representative sequences for the corresponding subtype from the GenBank database (for clade B, sequences B.US.83.RF, B.US.90.WEAU160, B.FR.83.HXB2, B.US.86.JRFL; for clades A-E, sequences A1.KE.93.Q23-17, A1.SE.94.SE7253, A1.UG.92.92UG037, A1.UG.85.U455; and for clade D, sequences D.CD.83.ELI.K03454, D.CM.01.01CM4412HAL.AY371157, D.UG.94.94UG114.U88824, and D.TZ.01.01TZA280.AY253311). The scale bar (horizontal line) indicates the number of substitutions per site.