Short Communication: Severe Immune Suppression in Patients Infected with R5-Tropic HIV-1 Strains Is Associated with Increased gp120 Net Charge at Variable Regions

Abstract

CXCR4-tropic viruses have been associated with advanced immune suppression. However, 50% of patients with AIDS exclusively harbor CCR5-tropic viruses. The net charge at HIV-1 envelope gp120 variable regions was examined in 66 HIV-1-infected individuals with CCR5-tropic viruses, of whom 30 had less than 200 cells/mm³. A positive net charge at gp120 variable regions was significantly associated with lower CD4 counts. Thus, the net charge at gp120 variable regions could influence HIV-1 disease progression in subjects with CCR5-tropic viruses.

CCR5 and CXCR4 are the major coreceptors used by HIV-1 to enter host cells. Based on the ability to use one or the other coreceptor, HIV-1 variants are classified as R5-tropic, X4-tropic, or dual-tropic. R5-tropic viruses predominate at early stages of HIV-1 infection, while the presence of X4-tropic variants has been associated with rapid declines in CD4 and advanced disease stages.¹ However, about 50% of patients evolving to AIDS exclusively harbor R5-tropic viruses.^2,3 An enhanced viral fitness and reduced susceptibility to HIV-1 entry inhibitors have both been recognized in R5-tropic viruses isolated from patients with advanced stages of infection.^4,5 Genotypic markers for these more pathogenic R5 variants have not been well characterized. Herein, we assess the association between the net charge of gp120 variable regions and the immune status in a large series of HIV-1-infected individuals exclusively harboring HIV-1 R5-tropic viruses.

All consecutive HIV-1-infected individuals on regular follow-up at our clinic who underwent HIV-1 tropism testing in early 2010 and were found to be exclusively infected with R5 variants were identified. They were divided into two groups according to whether their CD4⁺ T cell counts were above or ≤200 cells/mm³. The net charge at the HIV-1 gp120 variable regions was compared between these two groups. Moreover, net charge values were directly correlated with CD4⁺ T cell counts in the whole study population.

All HIV-1 gp120 variable regions were amplified and sequenced using three independent nested polymerase chain reaction (PCR) procedures, spanning the regions V1–V2, V4–C4–V5, and V3. The V1–V2 region was amplified using EnvB (5′-AGA AAG AGC AGA AGA CAG TGG CAA TGA–3′) and E125 (5′-CAA TTT CTG GGT CCC CTC CTG AGG-3′) as outer primers and EV1 (5′-ACA CAT GCC TGT GTA CCC AC-3′) and ES7A (5′-GCT AGA CTG CCA TTT AAC AG-3′) as inner primers. The V4–C4–V5 region was amplified using E80 (5′-CCA ATT CCC ATA CAT TAT TGTG-3′) and E41IAS1 (5′-CGC CCA TAG TGC TTC CTG CTGC-3′) as outer primers and V3E (5′-ATA AGA CAA GCA CAT TGT AAC-3′) and E41EAS1 (5′-CTC TCT GCA CCA CTC TTC TC-3′) as inner primers. The V3 region was amplified using primers and conditions already described elsewhere.^6,7 All amplicons were directly sequenced in the ABI PRISM 3100 Genetic Analyzer using the ABI PRISM Rhodamine Therminator reaction kit (Applied Biosystems, Foster City, CA). Sequences were analyzed using Seqscape software v2.5 (Applied Biosystems, Foster City, CA), considering nucleotide mixtures when the second highest peak in the electropherogram was above 25%. All sequences with nucleotide mixtures were expanded into all possible amino acid permutations and subsequently considered for tropism determination and net charge assessment. The pol gene was amplified using a commercial assay, the Viroseq HIV-1 Genotyping System (Abbott Diagnostics, Madrid, Spain). HIV-1 subtyping was carried out by phylogenetic analyses of pol and env genes.

The genotypic interpretation of HIV-1 tropism was performed using PSSM_X4R5-8, an optimized webPSSM approach, which has shown particularly good sensitivity for the detection of X4 variants.⁸ Thus, this tool is highly specific for the exclusion of patients harboring X4-tropic variants.

Specimens were considered as harboring R5-tropic viruses only when all permutations excluded X4-tropic strains. For net charge estimations, lysine and arginine were considered as positive amino acids and scored as +1, while glutamic and aspartic acids were considered as negative and scored as −1. Histidine was not considered for the calculation of net charges, following the recommendation made by others.^5,9 In sequences with amino acid mixtures, the mean net charge was calculated and considered for subsequent analyses.

Mann–Whitney tests and linear regression analyses were performed for comparisons and correlations, respectively. All statistical tests were two-tailed and only p values below 0.05 were considered as significant. All statistical analyses were performed using the SPSS v17.0 software (SPSS Inc., Chicago, IL).

A total of 66 HIV-1-infected patients exclusively harboring R5 variants were included in the study, 63 (95.5%) infected with subtype B and three (4.5%) with non-B clades (2 A1 and 1 G). Overall, 30 of these patients had CD4⁺ T cell counts below 200 cells/mm³ [median (interquartile range): 71 (43–167) cells/mm³] while 36 had more than 200 cells/mm³ [379 (320–630) cells/mm³]. Subjects with low CD4⁺ T cell counts had significantly higher plasma HIV-RNA than those with high CD4 counts [4.98 (4–5.28) vs. 4.02 (3.63–4.60) log copies/ml; p=0.002]. Overall, 30 (45.5%) individuals were antiretroviral (ARV)-experienced but naive to entry inhibitors. ARV-experienced patients were more frequent in the subset of patients with CD4⁺ T cell counts below 200 cells/mm³ than in the rest (63.3% vs. 33.6%, respectively; p=0.022). No significant differences were found when comparing the duration of HIV-1 infection in patients with CD4 counts above or below 200 cells/mm³ [4 (2–16) vs. 12 (6.5–15) years; p=0.223].

Genotypic information for all HIV-1 gp120 variable regions could be obtained for 57 of 66 (86.4%) patients. Failures in the amplification process and/or sequence analyses occurred in the remaining samples. A total of 61 V1, 64 V2, 66 V3, 59 V4, and 63 V5 sequences could be obtained for further subsequent analyses. Table 1 depicts the net charge values for all gp120 variable regions according to CD4 counts in the study population. Higher net charge values were observed in V1, V2, and V5 gp120 variable regions in patients with CD4⁺ T cell counts ≤200 cells/mm³ compared to those with greater CD4 counts. However, significant differences were observed only for the V5 region (median, 0 vs. −1 charge unit; p=0.042). Moreover, the sum of the net charge of all but V3 variable regions was also significantly greater in patients with CD4 counts ≤200 cells/mm³ than in the rest (median −1 vs. −3 charge units; p=0.032).

Table 1.

Net Charge of Variable Regions Within gp120 in R5 Strains from HIV-1-Infected Patients According to CD4 ⁺ T Cell Counts

	Net charge
	V1	V2	V3	V4	V5	All	All but V3
No. of sequences analyzed	61	64	66	59	63	57	57
Patients with CD4⁺
T cell counts ≤200 cells/μl (n=30)	−0.5 [−2–1]	1 [−1–1.5]	3 [2–3.6]	−1.5 [−2–0]	0 [−1–0]	−1.75 [−0.1–2.9]	−1 [−3.1–0]
Patients with CD4⁺
T cell counts >200 cells/μl (n=36)	−1 [−2–0]	0 [0–1]	3 [3–4]	−1 [−2.8–0]	−1 [−1–0]	1 [−1–2.5]	−3 [−4–1]
p value	0.122	0.485	0.126	0.859	0.042	0.141	0.032

Values are expressed as medians and interquartile ranges. Significant p values are highlighted in bold.

Linear regression analyses showed significant associations between CD4⁺ T cell counts and net charge at the V1 loop [β: −53.5; 95% confidence interval (CI): −104.3 to −2.7; p=0.039] and for net charge of the sum of all but V3 variable regions (β: −26.4; 95% CI: −52.3 to −0.5; p=0.046). In this analysis, β represented the change in CD4⁺ T cell counts per each charge unit gained in the corresponding variable region.

The molecular mechanisms underlying these findings might be those previously reported by Repits et al. ^4,5 The electrostatic interactions occurring during the viral entry process might explain an increase of the net charge at the gp120 variable regions in advanced stages of HIV-1 infection. During the approximation of HIV-1 particles to host cells, electrostatic repulsion may occur as a result of the interaction between negatively charged viral and cellular surfaces. In vitro studies have suggested that the addition of the cationic polymer polybrene favors the attachment of HIV particles to the surface of target cells.¹⁰ Thus, an increase in the positivity of net charges in gp120 variable regions may occur to counteract the electrostatic repulsion between cellular and viral surfaces in late stages of HIV infection. The enhanced viral attachment resulting from nonspecific electrostatic forces at late stages of HIV infection in persons infected with R5-tropic viruses could be responsible for a more rapid CD4⁺ T cell depletion in this subset of patients.

In summary, the results of our study highlight an association between the net charge at the HIV-1 envelope gp120 variable regions and the immune status of HIV-1-infected patients with R5-tropic viruses. Overall, a more positive net charge at the HIV-1 gp120 variable regions is more frequently seen in patients with lower CD4⁺ T cell counts. Thus, an increase in positive net charges might occur in an attempt for viral particles to overcome the electrostatic repulsion between cellular and viral surfaces in R5-tropic viruses.^4,5 Further longitudinal studies in patients infected with HIV-1 R5-tropic viruses would provide a better understanding of the mechanisms driving the evolution of net charges in the gp120 variable regions and establish its potential clinical relevance.

Footnotes

Acknowledgments

This work was supported in part by grants from Fundación Investigación y Educación en SIDA (FIES), the European NEAT project, Red de Investigación en SIDA (RIS, ISCIII-RETIC-RD06/006), and Fondo de Investigación Sanitaria (FIS, projects CP08/00214, CP0610284, PI06/01826, and FI09/00868).

Author Disclosure Statement

No competing financial interests exist.

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