R5X4 viruses are evolutionary, functional, and antigenic intermediates in the pathway of a simian-human immunodeficiency virus coreceptor switch

Abstract

To examine the pathway of the coreceptor switching of CCR5-using (R5) virus to CXCR4-using (X4) virus in simian-human immunodeficiency virus SHIV(SF162P3N)-infected rhesus macaque BR24, analysis was performed on variants present at 20 weeks postinfection, the time when the signature gp120 V3 loop sequence of the X4 switch variant was first detected by PCR. Unexpectedly, circulating and tissue variants with His/Ile instead of the signature X4 V3 His/Arg insertions predominated at this time point. Phylogenetic analysis of the sequences of the C2 conserved region to the V5 variable loop of the envelope (Env) protein showed that viruses bearing HI insertions represented evolutionary intermediates between the parental SHIV(SF162P3N) and the final X4 HR switch variant. Functional analyses demonstrated that the HI variants were phenotypic intermediates as well, capable of using both CCR5 and CXCR4 for entry. However, the R5X4 intermediate virus entered CCR5-expressing target cells less efficiently than the parental R5 strain and was more sensitive to both CCR5 and CXCR4 inhibitors than either the parental R5 or the final X4 virus. It was also more sensitive than the parental R5 virus to antibody neutralization, especially to agents directed against the CD4 binding site, but not as sensitive as the late X4 virus. Significantly, the V3 loop sequence that determined CXCR4 use also conferred soluble CD4 neutralization sensitivity. Collectively, the data illustrate that, similar to human immunodeficiency virus type 1 (HIV-1) infection in individuals, the evolution from CCR5 to CXCR4 usage in BR24 transitions through an intermediate phase with reduced virus entry and coreceptor usage efficiencies. The data further support a model linking an open envelope gp120 conformation, better CD4 binding, and expansion to CXCR4 usage.

FIG. 1.

(A) Comparison of V3 loop sequences of the parental R5 SHIV_SF162P3N isolate, the recovered X4 virus SHIV_BR24N, and viruses present in plasma and axillary (Ax) LN of macaque BR24 at 20 wpi. Dashes denote similarity in sequences, and gaps are indicated as dots. The net positive charge of this region is indicated in the right column. Positions 11 and 25 within the V3 loop that frequently distinguished HIV-1 X4 and R5 viruses (16) are designated by arrowheads, and the highly conserved PNG site at the V3 base is boxed. The numbers in parentheses represent the number of clones with the indicated V3 loop sequence reported relative to the total number of clones sequenced. (B) Representation of SHIV viral variants without (WT) or with HI or HR insertions in various tissue sites of macaque BR24 at the time of death (28 wpi). Percentages of Env clones present in axillary (axil), iliac (ili), inguinal (ing), colonic (col), and mesenteric (mes) LNs, as well as intraepithelial lymphocytes (IEL) and LPL of the gut with the indicated signature V3 loop sequences are shown. Numbers in parenthesis indicate the number of gp120 clones sequenced from each of the tissue sites.

FIG. 2.

(A) Phylogenetic tree showing the relationship between the Env V1 to V5 sequences of the parental R5 SHIV_162P3N, the week 20 variants, and the final X4 SHIV_BR24N. A neighbor-joining tree was generated, with the SHIV_SF162P3 sequence serving as an outgroup. Week 20 plasma sequences are marked in red while those of the X4 SHIV_BR24N isolate are in blue. The scale bar indicates genetic distances along the horizontal branches. The values on the branches represent the percentage of bootstrapped trees out of 1,000 replicates. Sequence gaps are excluded for analysis. (B) Comparison of the predicted V2, V3, C4, and V5 gp120 amino acid sequences of SHIV_SF162P3N, SHIV_BR24N, and week 20 Envs. The nine amino acids in X4 SHIV_BR24N that differed from R5 SHIV_SF162P3N Env gp120 are indicated in red. Dashes denote similarity in sequence, dots indicate gaps, and the PNG sites are boxed. Residues that differ between the viruses are numbered based on their relative positions in the HXB2 sequence. w, week.

FIG. 3.

Coreceptor usage of predominant week 20 HI insertion-bearing viruses. (A) CCR5 and CXCR4 usage of viruses pseudotyped with Env of SHIV_SF162P3N (P3N), SHIV_BR24N (BR24N), and week 20 viruses bearing HI insertions (HI₂₀) was determined by entry inhibition into TZM-bl CCR5⁺ CXCR4⁺ (TZM) cells with 1 μM CXCR4 inhibitor (AMD3100) or CCR5 inhibitors (TAK-779 and PSC-RANTES). Infection and blocking with cognate coreceptor inhibitors were also performed in the U87.CD4.CCR5 (B) and U87.CD4.CXCR4 (C) cells. Error bars indicate standard errors of the mean of data in triplicate wells. Results shown are representative of at least two independent experiments.

FIG. 4.

Entry and coreceptor usage efficiencies of R5, R5X4, and X4 SHIV viruses. (A) Relative entry into U87.CD4 (closed bars), U87.CD4.CXCR4 (open bars), and U87.CD4.CCR5 (hatched bars) cells of luciferase reporter virus expressing Env of R5 SHIV_SF162P3N (P3N), X4 SHIV_BR24N (BR24N), R5X4 (HI₂₀), and V3 loop recombinant EnvP3N(HI-V3). (B) Blocking of P3N (•) and R5X4 (□) virus entry into U87.CD4.CCR5 cells with increasing concentrations of the CCR5 inhibitor PSC-RANTES. (C) Blocking of BR24N (▵) and R5X4 (□) virus entry into U87.CD4.CXCR4 cells with increasing concentrations of the CXCR4 inhibitor AMD3100. Data shown are means ± standard errors of the mean of at least three independent experiments. RLU, relative light units.

FIG. 5.

Neutralization sensitivity of R5, R5X4, and X4 SHIVs. The neutralization susceptibility of the parental R5 SHIV_SF162P3N (•), the final X4 SHIV_BR24N (▵), and the R5X4 intermediate (□) virus to antibodies in serum from R5 SHIV_SF162P3N-infected (R5-SHIV serum) and X4 SHIV_BR24N-infected (X4-SHIV serum) macaques (A) and to soluble CD4 (CD4-IgG2) and MAbs IgG1b12, 447-52D, 2G12, 2F5, and 4E10 (B) was determined in TZM-bl cells as described in Materials and Methods. Data shown are the means and standard errors of at least three independent neutralization experiments.