Bioinformatic analysis of HIV-1 entry and pathogenesis

Abstract

The evolution of human immunodeficiency virus type 1 (HIV-1) with respect to co-receptor utilization has been shown to be relevant to HIV-1 pathogenesis and disease. The CCR5-utilizing (R5) virus has been shown to be important in the very early stages of transmission and highly prevalent during asymptomatic infection and chronic disease. In addition, the R5 virus has been proposed to be involved in neuroinvasion and central nervous system (CNS) disease. In contrast, the CXCR4-utilizing (X4) virus is more prevalent during the course of disease progression and concurrent with the loss of CD4(+) T cells. The dual-tropic virus is able to utilize both co-receptors (CXCR4 and CCR5) and has been thought to represent an intermediate transitional virus that possesses properties of both X4 and R5 viruses that can be encountered at many stages of disease. The use of computational tools and bioinformatic approaches in the prediction of HIV-1 co-receptor usage has been growing in importance with respect to understanding HIV-1 pathogenesis and disease, developing diagnostic tools, and improving the efficacy of therapeutic strategies focused on blocking viral entry. Current strategies have enhanced the sensitivity, specificity, and reproducibility relative to the prediction of co-receptor use; however, these technologies need to be improved with respect to their efficient and accurate use across the HIV-1 subtypes. The most effective approach may center on the combined use of different algorithms involving sequences within and outside of the env-V3 loop. This review focuses on the HIV-1 entry process and on co-receptor utilization, including bioinformatic tools utilized in the prediction of co-receptor usage. It also provides novel preliminary analyses for enabling identification of linkages between amino acids in V3 with other components of the HIV-1 genome and demonstrates that these linkages are different between X4 and R5 viruses.

Fig. 1. HIV-1 entry mechanism

HIV-1 entry has been shown to initially require the binding of trimeric gp120 to the host protein CD4 on the target cell plasma membrane. This interaction triggers a conformational change in the HIV-1 envelope protein that results in the enhanced exposure of the gp120 V3 loop, which was initially concealed by the V1/V2 region. The V3 loop subsequently engages one of two chemokine co-receptors, CXCR4 (left) or CCR5 (right). The overall charge of the V3 loop largely determines co-receptor usage, with CXCR4-utilizing virus having a greater net positive charge. In addition, in CXCR4-utilizing viruses the V3 loop contacts primarily the N-terminus and ECL-2 of CXCR4, while CCR5-utilzing viruses contact the N-terminus and ECL-1 of CCR5. Finally, co-receptor binding initiates the membrane fusion machinery of HIV-1 gp41.

Fig. 2. Structural and chemical composition of the V3 loop

The secondary structure and Van der Waals surface of the gp120 V3 loop (PDB ID 1CE4) is displayed using Jmol (left) [321, 322]. Helical structures are indicated in purple and pink and unstructured regions of the V3 loop are displayed in white. Representative peptide primary structures were generated using the PepDraw online tool (right) [323]. For CXCR4-utilizing V3, the HXB2 strain was used while for the CCR5-utilizing V3 the BaL strain was used. Charged side chains are indicated using boxes, with yellow indicating positively charged amino acids (R and K) and green indicating negatively charged amino acids (D and E). The white boxes represent any amino acid residue. The position of these residues is provided based on the alignment of the sequences against consensus subtype B sequence from LANL. The location of these residues is also highlighted in the three-dimensional structure of the V3 loop. In addition, positions 11 and 25 are indicated in a box due to their prominence in in silico prediction of co-receptor utilization.

Fig. 3. Overview of HIV-1 envelope co-receptor utilization evolution in periphery and CNS

A. Transmitted HIV-1 quasispecies infect CD4+ T cells and cells of the monocyte-macrophage lineage within the periphery (1). Following initial infection, a genetic bottleneck results in a clonal population predominantly exhibiting CCR5 co-receptor utilization (2). The founder swarm traffics to the CNS early in infection via infected monocytes (3) and can also traverse the blood-brain barrier as free virus (3). The brain founder swarm (4) is considered to be R5-utilizing and M-tropic on the basis of the cellular milieu within the CNS. Within the CNS, microglia and perivascular macrophages predominantly produce new virus while astrocytes are also infected, however this infection results in very limited virus production (5). Throughout chronic infection, virus may continue to traffic between physiological compartments (7). The trafficking between the periphery and CNS likely increases in magnitude over time (8) due to the leakiness of the blood-brain barrier during advanced neurological disease. Within the periphery, CD4+ T cells remain the predominant infected cell type and driver of viral evolution. As HIV-1 disease progresses, the emergence of X4- and X4/R5-using quasispecies is commonly observed (9). In late infection, many patients are observed to experience a tropism switch to a predominantly X4-utilizing swarm (10), however this switch is not universally observed and many patients remain predominantly R5-utilizing (11). B. Macrophage tropism is considered to occupy a spectrum of HIV-1 envelope genotypes and phenotypes that are predominantly CCR5-utilizing but also include dual-tropic and CXCR4-utilizing species. This differs from T-cell tropism in that most isolates, regardless of co-receptor usage, should be able to efficiently infect T cells. The proportion of T cells infected may vary based on co-receptor expression, however a robust infection should still develop in a majority of infected individuals.

Fig. 4. Identification of amino acid positions that co-selected with V3 in subtype B and C

Arcs between V3 positions and gp120, gp41, Nef, and the LTR represent co-evolving residues as defined by statistically significant mutual information (MI). Each row represents the subset of LANL sequences that were subtype B, subtype C, or either subtype B or C. Each column represents the subset of sequences that are CCR5 utilizing (R5), CXCR4 utilizing (X4), or either X4 or R5 utilizing as predicted by WebPSSM. The combined column shows the superimposed links with R5 in red, X4 in blue, and all-patients in black. Subsets that had fewer than 10 X4 or 10 R5 sequences of co-linear sequence were excluded from the analysis and the region was removed from the diagram.

Fig. 5. Differential amino acid distribution (Circos diagram)

Arcs between V3 positions and gp120, gp41, Nef, and the LTR that have differential residue preference as calculated by a Fisher’s Exact test. Each Circos diagram represents a subset of LANL sequences that were subtype B alone, subtype C alone, or either combination of subtype B and C. The combined figure shows the superimposed links with subtype B in green, subtype C in red, and subtype B and C in blue. Subsets that had fewer than 10 X4 or 10 R5 sequences of co-linear sequence were excluded from the analysis and the region was removed from the diagram.