Substitution of gp120 C4 region compensates for V3 loss-of-fitness mutations in HIV-1 CRF01_AE co-receptor switching

Abstract

HIV-1 infection is mediated by a viral envelope subsequently binding to CD4 receptor and two main coreceptors, CCR5 (R5) for primary infection and CXCR4 (X4) in chronic infection. Switching from R5 to X4 tropism in HIV-1 infection is associated with increased viral pathogenesis and disease progression. The coreceptor switching is mainly due to variations in the V3 loop, while the mechanism needs to be further elucidated. We systematically studied the determinant for HIV-1 coreceptor switching by substitution of the genes from one R5 and one X4 pseudoviruses. The study results in successfully constructing two panels of chimeric viruses of R5 to X4 forward and X4 to R5 reverse switching. The determinants for tropism switching are the combined substitution of the V3 loop and C4 region of the HIV-1 envelope. The possible mechanism of the tropism switching includes two components, the V3 loop to enable the viral envelope binding to the newly switched coreceptor and the C4 region, to compensate for the loss of fitness caused by deleterious V3 loop mutations to maintain the overall viral viability. The combined C4 and V3 substitution showed at least an eightfold increase in replication activity compared with the pseudovirus with only V3 loop substitution. The site-directed mutations of N425R and S440-I442 with charged amino acids could especially increase viral activity. This study could facilitate HIV-1 phenotype surveillance and select right entry inhibitor, CCR5 or CXCR4 antagonists, for antiviral therapy.

Keywords: C4 region; HIV-1; V3 loop; coreceptor-switching; tropism.

Figure 1.

Determination of cell tropism for the pseudovirus 167 and 248. (A) IC₅₀ of pseudovirus 248, 167, and 194 in a single-cycle infection assay was displayed in TZM-bl cells. The pseudovirus 248 is strongly inhibited by CXCR4 inhibitor Plerixafor (IC₅₀ of 0.69 nM) and weakly inhibited by CCR5 inhibitor Maraviroc. The pseudovirus 167 is strongly inhibited by CCR5 inhibitor Maraviroc (IC₅₀ 1.8 nM) and cannot be inhibited by Plerixafor. Pseudovirus 194 could be inhibited by Maraviroc and Plerixafor with the IC₅₀ of 1.85 nM and 9.68 nM respectively. (B) The inhibitory effect in Ghost-CD4 cell lines of two coreceptor antagonists on p167 and p248 at concentrations of 2 pm, 2 nm and 2 μm, respectively. (C) The pseudovirus p248 induced obvious GFP expression in the Ghost-CD4-CXCR4 cell line. The background level of fluorescence is weakly blocked by the CCR5 inhibitor Maraviroc (2 µM) but can be almost completely blocked by 2 µM of CXCR4 inhibitor Plerixafor, and the effect of Plerixafor-mediated inhibition was dose dependent.

Figure 2.

Entry efficiency and tropism of chimeric pseudovirus mutants in a single-cycle infection assay employing TZM-bl cell and Ghost-CD4-CCR5/Ghost-CD4-CXCR4 cells. (A) TCID₅₀ and the percentage of fluorescent positive cells are further detailed. (B) Pseudoviruses that mediate entry via CCR5 are shown in blue, pseudoviruses that mediate entry via both CCR5 and CXCR4 but more efficiently via CXCR4 are shown in red, and pseudoviruses that can’t detect fluorescence by either Ghost-CD4-CCR5 or Ghost-CD4-CXCR4 are shown in yellow.

Figure 3.

Eight different amino acids in C4 and its mutants. (A) The eight different amino acids in the C4 region of p167 and p248 are located at positions 419, 424, 425, 429, 430, 440, 442, 446, respectively (numbering based on HxB2). (B) Twenty-one mutants were designed with these eight amino acids and performed in p167-248V3 and p218-167V3, TCID₅₀ and the percentage of fluorescent positive cells is further detailed. TCID₅₀ is determined with TZM-bl cell. X4-tropic viruses are shown in red and R5-tropic viruses are shown in blue. Chimeras C1, C3, C22, and C23 are the control pseudoviruses. N425R showed the most significant enhancement of viral activity among the eight single-site mutations and highlighted in red colour in the table.

Figure 4.

Structural modelling of chemokine receptor binding to key amino acid of C4. An overview of modelling complex of CD4-gp120-CXCR4 is in the middle of this figure. CD4 is shown in green, coreceptor shown in blue, and the gp120 of pseudovirus 167-248V3 (C1) is shown in grey. Critical C4 positions 425 and 440–442 coloured in magenta and yellow, respectively. The mutagenesis of N425R is indicated on the left. Three contacts with the distance of 2.6, 2.8, and 2.9 Å exist between the Asn425 and the Phe43 of CD4. Upon mutation of asparagine to arginine, the contacts between the two amino acids changed from 3 to a closer 2.3 Å one. The electrostatic potential energy of the model of pseudovirus C1 to CXCR4 is shown on the right side of the figure. The introduction of the mutation S440D creates a negatively charged pocket in the N-terminus of CXCR4. Positive charged amino acids are in blue and negative in red.