Effect of chloroquine on reducing HIV-1 replication in vitro and the DC-SIGN mediated transfer of virus to CD4+ T-lymphocytes

Abstract

Background: Chloroquine (CQ) has been shown to inhibit HIV-1 replication in vitro as well as in vivo and has been proposed to alter the glycosylation pattern of the gp120 envelope. These activities indicate that the compound can be used not only as an effective HIV-1 therapeutic agent but also as a modulator of the gp120 envelope protein structure enabling for the production of broader neutralizing Ab responses.

Results: We confirm here that HIV-1 replication on CD4+ T-lymphocytes can be reduced in the presence of CQ and show that the reduced replication is producer cell mediated, with viruses generated in the presence of CQ not being inhibited for subsequent infectivity and replication. By analysing the gp120 envelope protein sequences from viruses cultured long-term in the absence or presence of CQ we demonstrate variant evolution patterns. One noticeable change is the reduction in the number of potential N-linked glycosylation sites in the V3 region as well as within the 2G12 Ab binding and neutralization epitope. We also demonstrate that HIV-1 produced in the presence of CQ has a reduced capacity for transfer by Raji-DC-SIGN cells to CD4+ T-lymphocytes, indicating another means whereby virus transmission or replication may be reduced in vivo.

Conclusion: These results indicate that CQ should be considered as an HIV-1 therapeutic agent with its influence exerted through a number of mechanisms in vivo, including modulation of the gp120 structure.

Figure 1

Viral replication in the presence of CQ. A) JR-CSF (R5) virus B) LAI (X4) virus replication was monitored in the presence of 200 μM, 100 μM, 50 μM of CQ or in the absence of CQ. Viral input for the replication assay was 100 TCID₅₀/ml with the CA-p24 concentration determined during the course of the infection.

Figure 2

Viral replication of C33A produced viruses in the presence of CQ. A) JR-CSF (R5) replication, B) 299.10 (R5X4) replication and C) LAI (X4) replication. All three viruses were produced by transfection of C33A cells pre-treated with 100 μM CQ or in its absence as a control. The replication capacity of the produced viruses were determined on CD4⁺ T-lymphocytes in the absence of CQ. CA-p24 at 1 ng/ml was used as viral input with the CA-p24 concentration determined during the course of the infection. Standard deviations are depicted in all panels. All replications were performed in triplicate.

Figure 3

Prolonged passage of HIV-1 in the presence of CQ. An R5X4 virus (293.10) was cultured for 206 days in the presence or absence of CQ (100 μM). The concentration of CA-p24 was determined in culture supernatants on either day 7 or 10 of culture and 15 ng/ml CA-p24 was added to fresh CD4⁺ T-lymphocytes. The culture was monitored for CA-p24 and the culture in the absence of CQ is depicted with a solid line and the culture in the presence of CQ is depicted as a broken line.

Figure 4

Replication of CQ passaged virus. The replication of CQ passaged or the control passaged 293.10 viruses were tested for their replication in the absence of CQ. CA-p24 or 1 ng/ml was used as input for monitoring replication A) day 37 of passage, B) day 77 of passage, C) day 103 of passage, D) day 147 of passage, E) day 183 of passage and F) day 206 of passage. Standard deviations are depict in all panels. All virus replications were performed in triplicate. G) Determination of TCID₅₀/ml values of passaged viruses in the absence or presence of CQ. Viral infectivity of the viruses passaged in the absence or presence of CQ (days 37, 77, 103, 147, 183 and 206) was measured on CD4⁺ T-lymphocytes. Standard deviations are depicted.

Figure 5

Sequence analysis of passages viruses in the presence or absence of CQ. HIV-1 RNA was isolated from culture supernatant and viral RNA was converted to cDNA and then subjected to a nested PCR in order to amplify a fragment covering the V1V2 – C4 region of the gp120 gene. Sequence analysis was performed on several clones of the CQ and control passages. The sequence of the original virus 293.10 is shown. A) the V1V2 region. B) the V3 region including the PNG site at the base of the loop. C) the V4 region. The black lines above the original sequence represent PNG sites.

Figure 6

DC-SIGN mediated transfer of CQ passaged viruses and C33A derived viruses in the presence of CQ. Raji and Raji-DC-SIGN cells were incubated with viruses before washing with PBS and addition of CD4⁺ T-lymphocytes. CA-p24 levels were determined at day 7 by standard ELISA. The CA-p24 levels of transfer by Raji cells alone were subtracted from the CA-p24 values of transfer observed with Raji-DC-SIGN cells. A) DC-SIGN dependent transfer of viruses cultured long-term in the presence or absence of CQ (days 14, 77 and 197). B) DC-SIGN dependent transfer of JR-CSF, 299.10 and LAI virus produced in C33A cells either in the presence or absence of CQ. Standard deviations are depicted in both panels and P-values given.