Evolution of the HIV-1 env gene in the Rag2-/- gammaC-/- humanized mouse model

Abstract

Rag2(-/-) gamma(C)(-/-) mice transplanted with human hematopoietic stem cells (DKO-hu-HSC mice) mimic aspects of human infection with human immunodeficiency virus type 1 (HIV-1), including sustained viral replication and CD4(+) T-cell decline. However, the extent of HIV-1 evolution during long-term infection in these humanized mice, a key feature of the natural infection, has not been assessed fully. In this study, we examined the types of genotypic and phenotypic changes in the viral env gene that occur in the viral populations of DKO-hu-HSC mice infected with the CCR5-tropic isolate HIV-1(JRCSF) for up to 44 weeks. The mean rate of divergence of viral populations in mice was similar to that observed in a cohort of humans during a similar period of infection. Many amino acid substitutions were common across mice, including losses of N-linked glycosylation sites and substitutions in the CD4 binding site and in CD4-induced epitopes, indicating common selective pressures between mice. In addition, env variants evolved sensitivity to antibodies directed at V3, suggesting a more open conformation for Env. This phenotypic change was associated with increased CD4 binding efficiency and was attributed to specific amino acid substitutions. In one mouse, env variants emerged that exhibited a CXCR4-tropic phenotype. These sequences were compartmentalized in the mesenteric lymph node. In summary, viral populations in these mice exhibited dynamic behavior that included sequence evolution, compartmentalization, and the appearance of distinct phenotypic changes. Thus, humanized mice offer a useful model for studying evolutionary processes of HIV-1 in a complex host environment.

FIG. 1.

Plasma viral loads over 44 weeks of infection for mice 5 to 8. The DKO-hu-HSC mice were infected intravenously with HIV-1_JRCSF. HIV-1 viremia in plasma samples (copies/ml) from all infected DKO-hu-HSC mice were measured at 3, 10, 22, 40, and 44 weeks postinfection for mice 5 to 8 and at 4 and 22 weeks postinfection for mice 64 and 67.

FIG. 2.

Phylogeny of HIV-1 sequences from all mice. The tree is a maximum likelihood tree of V1-to-C5 sequences from the peripheral blood at week 44 for mice 5 to 8 and at week 22 for mice 64 and 67. Symbols at branch tips indicate the mouse number. Open square, mouse 5; open triangle, mouse 6; closed triangle, mouse 7; diamond, mouse 8; closed circle, mouse 64; closed square, mouse 67. The open circle at the top of the tree indicates the JRCSF input sequence. Asterisks indicate bootstrap values of >70%. The scale bar indicates the number of substitutions per site.

FIG. 3.

Comparison of divergence rates and diversity levels in mice and humans. Distance measurements were based on branch lengths of maximum likelihood trees. Symbols for the mouse group in panels A and B indicate the mouse number. Open square, mouse 5; open triangle, mouse 6; closed triangle, mouse 7; diamond, mouse 8; closed circle, mouse 64; closed square, mouse 67. (A) Divergence rates in mice and in human cohorts initially sampled postseroconversion (human postseroconversion) (86) and preseroconversion (human pre-to-postseroconversion) (83) were measured for the C2-to-V5 region of env. For the postseroconversion data set, the mean distance, based on branch lengths of a maximum likelihood tree, was measured from the consensus at a time point 2 to 5 months postseroconversion for variants sampled at time points 9 to 24 months later. Sequences sampled at the first time point were relatively homogeneous, except for two subjects who were likely infected with two variants and were removed from the analysis. For the pre-to-postseroconversion data set, mean distance was measured from the consensus at time points estimated to be 20 to 24 days postinfection and 9 to 16 days prior to seroconversion for variants sampled 3 to 19 months later. Mean distance was divided by the time between sampling points to obtain the rate of divergence. For mice, the mean distance was measured from HIV-1_JRCSF for variants sampled at the last time point, at either 22 or 44 weeks postinfection. (B) Population diversity is represented as the mean pairwise distances within populations at or extrapolated to 44 weeks postinfection, for mouse and pre-to-postseroconversion human samples, or postseroconversion for the postseroconversion human samples. The student t test was used to determine P values for differences between distributions. P values for all comparisons were >0.2.

FIG. 4.

Positions and classifications of amino acid substitutions in the V1-to-C5 region of Env. Amino acid numbering on the abscissa corresponds to the HXB2 reference sequence. Bars on the graph indicate positions of substitutions and the frequency with which substitutions were identified in a mouse population at the final time point postinfection. Variable loops are shaded in gray. Substitution types are indicated by symbols. Square, addition of a PNLG site; diamond, loss of PNLG site; open triangle, >95% sequence conservation in subtype B; closed circle, component of the CD4 binding domain; asterisk, reversion to subtype B consensus; closed triangle, linked to coreceptor tropism. Substitutions at positions 165 and 167 that affected Env conformation and substitutions identified in three or more mice are indicated along with their mean percent abundances in the population. Substitutions identified in both donor sets are indicated by “AB” (see Table 1). “TC” indicates substitutions associated with tissue culture adaptation in other studies (see Discussion). Assessment of >95% sequence conservation was based on a random sampling of 300 sequences (one per patient) from the Los Alamos HIV Sequence Database. Sequencing of 30 SGA amplicons from the inoculum revealed four single point mutations among three amplicons. One of these mutations, at codon 151, was observed later in infection, in mouse 67.

FIG. 5.

Neutralization sensitivities of selected envelope clones and mutants to 447-52D. (A) Clone names give the mouse number followed by the clone number (e.g., 5_10). Percent inhibition is indicated for 447-52D antibody concentrations of 10 to 0.1 μg/ml. Error bars represent the ranges for two replicates. SF162c, a neutralization-sensitive chimeric env gene in which the V3 region of HIV-1_SF162 was replaced with the V3 region from HIV-1_JRFL (71). (B) Alignment of the V2 loops of clones tested for neutralization sensitivity to 447-52D. Positions 165 and 167 are indicated. (C) Neutralization sensitivity of HIV-1_JRCSF with I165R, D167N, or I165K substitution alone.

FIG. 6.

Phenotypic analysis of coreceptor tropism. (A) Relative CXCR4 and CCR5 activities of env clones from four mice at week 44 postinfection in a pseudotype entry assay using U87.CD4 cells expressing either CXCR4 or CCR5. env clones exhibiting X4 usage are boxed and labeled “dual tropic.” Error bars represent the standard deviations for three replicates. These data are representative of multiple tropism assays. (B) Alignment of the V3 loops of env clones tested for panel A. Clone names indicate the mouse number, tissue origin (peripheral blood [PB]), and clone number. Env proteins exhibiting X4 tropism, boxed in panel A and indicated as “dual tropic,” are indicated in the alignment as those with changes in their V3 loops at positions 11 and 25 associated with the ability to use CXCR4. These variants were identified only in mouse 6 and comprised 15% of the population.

FIG. 7.

Maximum likelihood phylogeny of env variants (V1 to C5) recovered from different tissue compartments in mouse 6. Bootstrap values of >80% and >50% are indicated by double and single asterisks at nodes, respectively. Sequences were derived from viral DNA except in the case of sequences from the peripheral blood, which were from plasma viral RNA. The tissues of origin of sequences are indicated by symbols. Triangles, spleen; diamonds, bone marrow; squares, peripheral blood; closed circles, mesenteric lymph node. An open circle indicates the JRCSF input sequence. The boxed cluster indicates X4-like V3 sequences, which contain substitutions at positions 11 and 25, in addition to others, and cluster together. A subset of these sequences was phenotypically tested to confirm tropism. Of the variants in the MLN, 95% contained an X4-like V3 sequence, indicating compartmentalization of this phenotype in this tissue in mouse 6 (P < 0.0001).