To confirm that CQ has an inhibitory effect on the in vitro replication of HIV-1 we separately cultured an R5 (JR-CSF) and X4 (LAI) virus on CD4⁺ T-lymphocytes and monitored replication in the presence of variant concentrations of CQ (200, 100 and 50 μM). We observe that CQ inhibits the replication profile of both viruses in comparison to the control cells (Fig. 1). When comparing the dose dependent inhibitory effect of CQ on viral replication the R5 virus (Fig. 1A) appears more sensitive than the X4 virus (Fig. 1B), suggesting a co-receptor phenotype restriction to inhibition by CQ. The observed inhibition by CQ was not due to enhanced cell death since cell counts and viabilities were identical in the 100 and 200 μM CQ cultures to the non-CQ treated control cells during one week of culture (data not shown).

In order to determine whether the inhibitory effect of CQ was mediated through altered infectivity of generated virus particles we analyzed replication on CD4⁺ T-lymphocytes of HIV-1 produced in C33A cells pre-treated with 100 μM CQ. The viruses JR-CSF (R5), 299.10 (R5/X4) and LAI (X4) produced from cells not treated with CQ showed higher CA-p24 levels than viruses produced from cells treated with CQ (data not shown). When we studied the replication kinetics of the viruses away from CQ with a set CA-p24 viral input there was no difference in replication of the viruses generated in the presence or absence of CQ (Fig. 2). TCID₅₀/ml values were identical for all three viruses generated in the presence or absence of CQ (data not shown). These results indicate that viruses produced in the presence of CQ are as equally infectious as those produced in its absence and that the effect of the drug on lowering CA-p24 production is mediated at the cellular level.

To monitor the effect of long-term culturing of HIV-1 in the presence of CQ we passaged virus 293.10 (R5X4) for 30 weeks either in the absence or presence of CQ (100 μM). Each week, or when CA-p24 levels were sufficiently high, a set viral load (15 ng/ml) was transferred to fresh CD4⁺ T-lymphocytes and cultured. CA-p24 production was consistently lower for the virus passaged in the presence of CQ compared to the control passsaged virus (Fig. 3), even after 206 days of culture (30 passages). These results demonstrate that HIV-1 does not evolve to escape the inhibitory effects of CQ.

Since we have shown previously that viral replication was not altered after HIV-1 production on C33A cells in the presence of CQ we wished to identify whether this was the same for the long-term cultured virus stocks. The replication profile of harvested viruses from various time-points during the passage in the presence or absence of CQ was determined on CD4⁺T-lymphocytes (Fig. 4). The replication at day 37 showed an increase in replication for the CQ passaged virus population versus the non-CQ treated culture (Fig. 4A). On the contrary for day 77 (Fig. 4B), day 103 (Fig. 4C), day 147 (Fig. 4D), day 183 (Fig. 4E) and day 206 (Fig. 4F) no differences in replication between the CQ passaged viruses and the non-CQ passaged viruses were observed. These results indicate that there is no difference in replication of HIV-1 after long-term culture in the presence or absence of CQ, although the virus from the CQ treated day 37 culture showed an enhanced replication over the non-CQ treated stock. The fact that the later viruses did not show such an increase indicates that the result observed for the day 37 CQ passaged virus was most likely due to experimental variation and reflects the poor infectivity of the viruses from that time-point. However, the main finding is that CQ did not diminish the replication capacity of HIV-1. TCID_50/ml values were determined for stocks generated on days 37, 77, 103, 147, 183 and 206 during the prolonged passage in the absence or presence of CQ. Both culture conditions demonstrated an increased infectivity of virus over time (Fig. 4G), indicating that viruses in the presence of CQ adapt as efficiently as non-CQ treated cultures. This again reiterates that CQ exerts a cellular restriction to viral production and not a direct effect on viral infectivity.

A previous study has suggested that CQ can modify the PNG patterns of the gp120 envelope 2. We therefore wished to determine whether HIV-1 passaged in the presence of CQ had a similar gp120 envelope sequence to virus passaged in the absence of CQ. DNA sequence analysis of a number of cloned PCR products of gp120 identified that the overall amino acid charge of the V1V2 region (Fig. 5A) is significantly higher for the CQ passaged virus compared to the control passage (P = 0.001), or the original virus (P = 0.001) (Table 1). On the contrary, the overall positive charge of the gp120 V3 region (Fig. 5B) is significantly lower (P < 0.0001) in the CQ passaged virus but equal to the original virus (Table 1). A significant decrease in V4 region length (Fig. 5C) is also identified in the CQ passaged virus in comparison to the control (P < 0.0001), or the original 293.10 virus (Table 1). Of particular interest is the observation that the PNG profile of the V3 region (Fig. 5B) was significantly reduced after passage of the 293.10 virus in the presence of CQ with the virus reducing the number of PNG sites in V3 region from 2 to 0 (P < 0.0001), whilst in the non-CQ treated culture it is reduced from 2 to 1.7 (Table 1). Overall the sequence analysis reveals that there are differences in the envelope sequences of viruses cultured in the presence of CQ that may have an influence on the virus phenotype or the immunogenic properties of gp120.

We compared the gp120 sequences of the passaged viruses with what is known for the 2G12 binding site, a monoclonal Ab with broad neutralizing activity against HIV-1 subtype B isolates. This antibody has a known PNG component to its recognition epitope 23. For the virus passaged in the presence of CQ we observed a loss of two PNG sites (332 and 397) that have been shown to express carbohydrates important for 2G12 binding 23, as well as an additional site in the V3 region of gp120 (data not shown). The PNG site expressing carbohydrates involved in 2G12 binding (397) is lost in the V4 region due to a deletion of 5 amino acids. Loss of a PNG site in the V4 region is also observed in the control passage (Table 1) but does not involve this specific site under question since the deletion is 7 amino acids upstream from position 397.

Since PNG sites were altered in the CQ passaged viruses and these events are known to be involved with HIV-1 binding to DC-SIGN 2023 we tested the efficiency by which the viruses were transferred by Raji-DC-SIGN cells to CD4⁺ T-lymphocytes. When comparing the viruses produced on days 14, 77, 135 and 197 we observe that for viruses produced in the absence of CQ there is a significantly higher level of DC-SIGN mediated transfer than viruses produced in the presence of CQ (day 14, P = 0.001; day 77, P = 0.004; day 197, P = 0.005) (Fig. 6A). These results indicate that the alteration of the PNG sequence of gp120 may alter its binding to DC-SIGN, or alternatively the glycosylation machinery of the cell can influence the interaction of the virus with DC-SIGN. To test the latter we monitored the Raji-DC-SIGN mediated transfer to CD4⁺ T-lymphocytes of viruses produced on C33A cells (CQ or non-CQ treated). All three viruses were shown to have a reduced capacity for DC-SIGN mediated transfer when produced in cells treated with CQ over viruses generated in non-CQ treated cells (JR-CSF, P = 0.0009; 299.10, P = 0.002; LAI, P = 0.003) (Fig. 6B). This result indicates that the same virus produced in the presence of CQ has a reduced capacity for transfer by DC-SIGN expressing cells to CD4⁺ T-lymphocytes.

Raji and Raji-DC-SIGN cells were cultured and utilized as previously described 1037. Peripheral Blood Mononuclear Cells (PBMCs) were isolated from three buffy coats from different HIV-1 uninfected donors by standard Ficol-Hypaque density centrifugation, pooled and frozen in multiple vials. After thawing, PBMCs were activated with phytohemagglutinin (2 μg/ml) and cultured in RPMI medium containing 10% FCS, penicillin (100 units/ml) and streptomycin (100 μg/ml) with recombinant interleukin-2 (100 units/ml). On day 3 the cells underwent CD8⁺ cell depletion using CD8 immunomagnetic beads according to the manufacturers instructions and CD4⁺ T-lymphocytes were cultured with IL-2 (100 units/ml). The human carcinoma cell line C33A was cultured in DMEM, with 10% FCS, penicillin (100 units/ml) and streptomycin (100 μg/ml).

Replication competent HIV-1 stocks of JR-CSF (R5), LAI (X4) and 293.10 (R5X4) molecular cloned viruses (previously described in ref 38 were generated by passaging virus through isolated CD4⁺ T-lymphocytes. Virus stocks were also produced by transfection of C33A cells with plasmid expressing the specific virus strain to be analyzed and using the assay described below. The CA-p24 levels in the culture supernatants were determined using a standard ELISA protocol. Virus stocks were generated either in the presence (100 μM) or absence of CQ.

The R5X4 dual-tropic molecular cloned virus (293.10) was the starting virus for the passage and has been previously described 38. Fifteen ng/ml of CA-p24 was added to 10 × 10⁶ CD4⁺ T-lymphocytes in a volume of 5.0 ml RPMI medium with 100 μM CQ being added to the CQ passage culture flask. HIV-1 CA-p24 concentration was determined once a week with new virus being added to fresh CD4⁺ T-lymphocytes. If the CA-p24 levels were too low then the CA-p24 was re-determined 3 days later with 15 ng/ml subsequently added to fresh cells. Remaining culture supernatants and cell pellets after each passage were stored at -80°C until required for analysis.

TCID₅₀/ml values were determined by limiting dilution of the viral stock on CD4⁺ T-lymphocytes, as previously described 38. In short the CD4⁺ T-lymphocytes were plated at 2 × 10⁵ cells/well in 96 well plates with 5 fold serial dilution of the virus. On day 7 the wells were scored for CA-p24 levels and the number of positive wells determined. These values were used to determine the TCID₅₀/ml values for each virus. For the determination of the TCID₅₀/ml for the C33A generated viruses and the viruses after prolonged culture, the input was standardized at 105 ng/ml CA-p24 and 10.5 ng/ml CA-p24, respectively.

CD4⁺ T-lymphocytes were plated at 1 × 10⁵ cells/well in 96 well plates. One hundred TCID₅₀ of virus stock was utilized with CQ being added either at 200 μM, 100 μM or 50 μm/well. For replication analysis of the C33A generated viruses and the viruses obtained from the prolonged CQ passage 1 ng/ml of CA-p24 was utilized as virus input. When analyzing viruses obtained from CQ cultures the level of CQ in the culture supernatant was compensated for in the control culture or TCID₅₀/ml determination assay. CA-p24 values were determined using a standard ELISA assay for the culture supernatants obtained from the infection assay collected over time. All experiments were performed in triplicate with the standard means depict.

Transfection of C33A cells was performed with 10.0 μg of plasmid DNA expressing HIV-1 using the CaCl₂ precipitation method. All plasmid DNA used was prepared using Qiagen kits. The DNA precipitate was split between two wells of C33A cells plated 24 hours earlier at 1.5 X10⁶ cells/well in a 6 well tissue culture plate in DMEM medium either in the absence or presence of CQ (100 μM). The transfections were performed in a final concentration of 6 ml of DMEM, with penicillin (100 units/ml), streptomycin (100 μg/ml) and 10% FCS. The following day the cells were washed with PBS and fresh media was added, the viral stock was harvested on day 3 of culture with the viral CA-p24 levels determined by standard ELISA.

The assay was performed as previously described 37. The Raji and Raji-DC-SIGN cells were plated at a concentration of 2 × 10⁴ cells/well in a 96 well format. Four hundred ng/ml of the appropriate virus was added to the Raji-DC-SIGN or Raji cells when studying the C33A produced virus stocks. For the CQ passaged viruses a set CA-p24 input of virus was utilized for each virus set (range 100 – 400 ng/ml). For the CQ passaged viruses the presence of CQ in the supernatant was compensated for in the control virus stock with an equal concentration of CQ added. After 2 hr incubation the culture was washed with PBS before addition of CD4⁺ T-lymphocytes at a concentration of 1 × 10⁵ cells/well. CA-p24 values were determined on day 7 using a standard ELISA protocol and all experiments were performed in triplicate.

HIV-1 RNA was isolated from culture supernatant according to the method of Boom 39. Viral RNA was converted to cDNA and then subjected to a nested PCR to amplify a fragment covering the V1V2 to C4 region of the gp120 gene. DNA sequence alignments were performed manually. Positions containing an alignment gap were included for the pair-wise sequence analysis. Phylogenetic analysis of the amplified region was performed with the neighborhood-joining (N-J) analysis of MEGA 40. The PNG sites were analyzed using the program available at the HIV-1 sequence database 41.

All statistical comparisons were performed using ANOVA with P < 0.01 being considered as statistically significant.