1 Department of Laboratory Medicine, Division of Medical Microbiology, Lund University, Sölvegatan 23, 223 62 Lund, Sweden
2 Department of Experimental Medicine, Lund University, Sölvegatan 23, 223 62 Lund, Sweden
3 Venhälsan, Department of Infectious Medicine, Karolinska University Hospital, South Hospital, Stockholm, Sweden
4 Department of Virology, Swedish Institute for Infectious Disease Control, Karolinska Institute, Stockholm, Sweden
Correspondence
Marianne Jansson
marianne.jansson{at}med.lu.se
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession numbers for the sequences reported in this paper are DQ156998DQ157007.
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INTRODUCTION |
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The coreceptors utilized by HIV-1 include CCR5 and/or CXCR4, which are seven transmembrane chemokine receptors (Feng et al., 1996; Deng et al., 1996
; Dragic et al., 1996
; Cheng-Mayer et al., 1997
; Berger et al., 1999
). Early in HIV-1 infection, viruses primarily use CCR5 as a coreceptor (R5 phenotype) for host-cell entry (van't Wout et al., 1994
). Whether this is the result of a selective advantage at the time of virus transmission or when infection is established in the new host is yet to be determined. However, approximately half of the individuals infected with HIV-1 develop viruses at a later stage with the ability to use CXCR4 either solely (monotropic) or in combination with other chemokine receptors (dual-/multitropic) (Bjorndal et al., 1997
). The appearance of viruses using CXCR4 is correlated with an increased virulence and more aggressive disease progression (Koot et al., 1992
; Karlsson et al., 1994
; Connor et al., 1997
; Fenyö, 2001
). Nevertheless, individuals who do not switch viral phenotype, and thus maintain an R5 virus phenotype throughout the course of the disease, eventually develop AIDS (de Roda Husman et al., 1999
; Jansson et al., 1999
). The natural CCR5 ligands RANTES, MIP-1
and MIP-1
can inhibit the replication of R5 viruses, whereas viruses using CXCR4 are resistant to inhibition by these chemokines (Cocchi et al., 1995
; Jansson et al., 1996
). However, our previous studies of patients who maintain the R5 virus phenotype throughout the course of the disease showed that R5 virus variants with reduced sensitivity to inhibition by RANTES may appear after AIDS onset (Jansson et al., 1996
, 1999
). Entry inhibitors target the initial binding step between HIV and the target cell, and accordingly inhibit the fusion of the cellular and viral membranes (LaBranche et al., 2001
; Moore & Doms, 2003
). The recent appearance of multidrug-resistant HIV-1 variants has triggered the research and development of alternative antiretroviral agents, including entry inhibitors. Thus, the establishment of the baseline susceptibility to entry inhibitors of HIV-1 variants emerging during disease progression may be important in the optimal design of such treatments.
With this in mind, we investigated the sensitivity to the entry inhibitors T-20 and TAK-779 of R5 viruses from patients who maintain the R5 phenotype during the course of the disease but develop R5 virus variants with reduced RANTES sensitivity after AIDS onset. Furthermore, additional biological properties, such as the infectivity and replicative capacity of these R5 isolates, were analysed.
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METHODS |
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U87.CD4-CCR5 infection assay.
U87.CD4-CCR5 cells (Bjorndal et al., 1997) were cultured in complete DMEM medium (Invitrogen) supplemented by 10 % FCS, 0·1 µg streptomycin ml1 and 0·1 U penicillin ml1. Cells were seeded (0·5 ml) in 48-well plates and incubated overnight, or until they reached 5060 % confluence. Infection was performed as described previously (Shi et al., 2002
), with the exception that inoculum virus (8·5 ng RT ml1) was RT-normalized and serially diluted in fivefold steps. On day 5, the cells were fixed in a methanol/acetone (1 : 1) mixture and stained with haematoxylin (Merck) to visualize the syncytia (plaque formation). The number of p.f.u. per well was determined by microscopic analysis.
Entry inhibitor sensitivity assay.
For the analysis of R5 virus sensitivity to entry inhibitors, PBMC from four donors were pooled and infected as described above, with the exception that RANTES (PeproTech EC Ltd, London, UK), T-20 and TAK-779 were added. Infections were performed with inoculum virus normalized both to RT concentration (0·33 ng RT ml1) and to TCID50 (40x). T-20 fusion inhibitor (Wild et al., 1994; Kilby et al., 1998
) from Roche and TAK-779 (Baba et al., 1999
; Dragic et al., 2000
; Takashima et al., 2001
) were obtained from the NIH Research and Reference Reagent Program, Division of AIDS, NIAID, NIH. Inhibitors were serially diluted in threefold steps, starting at the absolute concentrations of 45 nM T-20, 330 nM TAK-779 and 77 nM RANTES, and simultaneously added to the cells and virus. Control cultures without entry inhibitor were infected in parallel. Infected PBMC were washed with PBS on day 1 and fresh inhibitors at concentrations corresponding to the set-up were added to the cultures. Supernatants were harvested on days 4 and 7, and p24 antigen content was analysed by ELISA (BioMérieux). The sensitivity to entry inhibitors was evaluated as IC50 and calculated from p24 antigen release in the control cultures.
Sequencing of the gp41 HR1 region.
The gp41 HR1 region of the R5 isolates studied was sequenced directly by obtaining RNA from virus stocks using NucleoSpin columns (Macherey-Nagel) according to the manufacturer's protocol. RNA was then transcribed to cDNA by reverse transcriptase PCR using random hexaoligonucleotides, Superscript II and RNase out (Invitrogen). For amplification of the specific gp41 fragment, PCR with Pfx polymerase (Invitrogen), forward primer 5'-CTTGGGAGCAGCAGGAAGCACT-3' and reverse primer 5'-GGTGAGTATCCCTGCCTAACTCT-3' (Invitrogen), was used. The amplified DNA fragment was then purified with the QIAquick PCR purification kit (Qiagen) and sequenced using forward sequencing primer 5'-CAGCAGGAAGCACTATGGGCG-3', reverse sequencing primer 5'-TATCCCTGCCTAACTCTATTCACTA-3' and the BigDye sequencing kit (Applied Biosystems). Sequences were then separated and analysed using a 3100 Genetic Analyser Hitachi (Applied Biosystems). The gp41 HR1 env sequences from the ten R5 isolates, corresponding to amino acids 2180 of the HXB2 sequence, were aligned to 20 subtype B and two subtype D reference sequences from the Los Alamos database (http://www.hiv.lanl.gov/) using BioEdit software. Neighbour-joining phylogenetic trees were constructed from 516 unambiguously aligned and gap-stripped nucleotides using the MEGA version 3.0 software and the Kimura substitution model.
Statistical analysis.
For the calculation of statistics, Statistica version 7 software was used. The non-parametric Spearman's rank correlation was used for the analysis of correlations, whereas Wilcoxon's matched pairs test was used for comparing the viral properties of early and late R5 isolates.
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RESULTS |
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DISCUSSION |
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Both our work and that of others has demonstrated that also in patients who maintain the R5 viral phenotype throughout the disease course, viral evolution occurs in vivo (Jansson et al., 1996, 1999
; Koning et al., 2003
; Karlsson et al., 2004
). Our earlier findings have shown that R5 virus variants isolated after AIDS onset may display decreased sensitivity to RANTES inhibition (Jansson et al., 1996
, 1999
). This observation has recently been confirmed by Koning et al. (2003)
, who found that RANTES-resistant R5 virus variants appeared as the disease progressed. These findings suggest an evolution of viral properties towards enhanced viral fitness with respect to the earliest events in the infection cycle, such as receptor binding. In the present study we corroborate this hypothesis by demonstrating that R5 isolates with reduced RANTES sensitivity also show enhanced infectivity and replicative capacity.
Interestingly, we recently noted that R5 virus variants with an altered mode of coreceptor use, in the form of a broadened ability to use CCR5/CXCR4 chimeric receptors, correlated with reduced RANTES sensitivity and decreasing numbers of CD4+ T cells (Karlsson et al., 2004). The assumption that the mode of receptor use is altered is also supported by the observation that late R5 viruses did not benefit from cationic polymer assistance. This was an unexpected observation, since polybrene has previously been shown to enhance retroviral adsorption rate (Davis et al., 2002
), and thus warrants further investigation. The mechanism of action of the cationic polymer polybrene has been suggested to involve the non-specific equalizing of charge differences between the glycocalyx on the target cell and the viral membrane (Davis et al., 2002
). Thus, late R5 isolates appear to have developed stronger, specific receptor binding.
Further support for the evolution of R5 variants with altered receptor binding-properties is provided by our finding that R5 viruses obtained after AIDS onset may be less sensitive to inhibition by the entry inhibitors TAK-779 and T-20. TAK-779, a small-molecule CCR5 antagonist binding to a pocket between CCR5 transmembrane helixes 1, 2, 3 and 7, has previously been shown to exert selective anti-HIV activity towards R5 viruses (Baba et al., 1999; Dragic et al., 2000
; Takashima et al., 2001
). T-20, on the other hand, is a synthetic peptide corresponding to a gp41 segment that inhibits HIV of both R5 and X4 phenotypes by blocking the step preceding fusion of the viral and cellular membranes (Wild et al., 1994
; Kilby et al., 1998
). Thus, the development of R5 virus variants with reduced sensitivity to both CCR5 agonist and antagonist, RANTES and TAK-779, in addition to the fusion inhibitor T-20, suggests that these virus variants may have developed an altered ability to bind the HIV-1 receptors, by the modification of binding affinity, receptor binding site or fusion kinetics. Recently, it has been suggested by Reeves et al. (2002)
that sensitivity to blockade by T-20 and TAK-779 correlates with R5 virus binding affinity. High CCR5 affinity results in more rapid fusion kinetics. In an experimental system, it has also been shown that in vitro passage of R5 virus in the presence of AD101, another small-molecule CCR5 antagonist, selects for a highly resistant escape mutant still dependent on CCR5 for host-cell entry but with increased CCR5 affinity (Trkola et al., 2002
). Accordingly, HIV-1 evolution is not restricted to a switch in coreceptors, but may also include modifications in the utilization of the coreceptor currently employed.
It is known that the expression levels of -chemokines, being ligands of CCR5, are elevated in HIV-1-infected individuals (Clerici et al., 1996
; Ullum et al., 1998
). It is possible that CCR5 ligands may exert selection pressure for the development of R5 variants with improved binding properties due to down-modulation of CCR5. An altered cytokine milieu resulting in the reduced expression of CCR5 has also been reported to affect viral phenotype (Valentin et al., 1998
; Patterson et al., 1999
; Llano et al., 2001
). Alternatively, the lack of proper immune response during severe immunodeficiency may allow the evolution of HIV-1 variants with an altered biological phenotype. In line with this, co-infection with a mix of R5 and X4 simian/human immunodeficiency virus (SHIV) strains in a rhesus macaque model (Harouse et al., 2003
), suggested that X4 strains were preferentially suppressed in the immunocompetent host. On the other hand, since our results demonstrate that the number of CD4+ T cells at time of isolation is associated with altered biological properties of R5 viruses, it is tempting to speculate that selective pressure acts upon the virus to evolve new properties in order to be able to infect the limited numbers of target cells. The evolution of certain virus variants following the selective loss of particular CD4+ T-cell subsets has been reported (Blaak et al., 2000
), and reduced dependence on CCR5 and CD4 expression has also been suggested to be linked to R5 virus affinity and macrophage tropism (Gorry et al., 2002
).
In order to evaluate the infectivity, replicative capacity and sensitivity to entry inhibitors of different R5 variants, care was taken to minimize interference from viral binding properties. We chose to normalize the virus inoculum on the basis of functional RT, since RT activity has been shown to correlate more closely with the number of infectious viral particles than does the quantity of p24 antigen (Corrigan et al., 1998; Malmsten et al., 2003
; Marozsan et al., 2004
). This is because p24-capturing assays measure the presence of the p24 antigen (i.e. infectious, non-infectious and decaying viruses), whereas the RT assay measures the enzymic activity of the reverse transcriptase, which is rapidly lost outside an intact virus particle. Nevertheless, we chose to analyse sensitivity to entry inhibitors in two ways, by using both RT and TCID50 normalization of inoculum virus. Both of these analyses revealed that late R5 viruses, less sensitive to RANTES, also displayed reduced sensitivity to the entry inhibitors T-20 and TAK-779. Altered envelope incorporation onto virions has recently been reported to influence both SIV and HIV infectivity (Yuste et al., 2004
; Bachrach et al., 2005
). Even though our analysis demonstrated that early and late R5 viruses differ in entry-inhibitor sensitivity when inoculum virus is normalized on the basis of TCID50, we cannot exclude that variation in envelope incorporation may contribute to the observed differences in infectivity and replicative capacity. Thus, the quantification of envelope density on virions of early and late R5 isolates merits further investigation.
Treatment with T-20, also known as enfuvirtide, has revealed that resistant HIV-1 variants may emerge as the result of mutations within the HR1 region of gp41 (residues 3645) (Rimsky et al., 1998; Wei et al., 2002
; Greenberg et al., 2004
). The R5 isolates used in our study were all derived from patients who were naïve to T-20 treatment, and sequence analysis revealed that none of the early or late isolates had acquired any of the mutations within the gp41 HR1 region previously linked to T-20 resistance. However, another study on the characterization of baseline susceptibility to T-20 has also suggested that variation among primary HIV-1 isolates from patients naïve to entry-inhibitor treatment may exist without polymorphisms in the HR1 region of gp41 (Labrosse et al., 2003
). This is in line with our observations, yet our results reinforce that the variability in sensitivity to T-20 inhibition of R5 HIV-1 variants is associated with disease progression. In contrast to Labrosse et al. (2003)
, who did not investigate sequential isolates obtained before and after AIDS onset, we found that the sensitivity to RANTES inhibition of the R5 isolates studied correlated with sensitivity to T-20. Our previous analysis excluded sequence variation within the gp120 V3 region as a determinant for phenotypic differences between early and late R5 isolates (Jansson et al., 1999
). Taken together, these results suggest that determinants that account for the natural emergence of R5 variants of lower sensitivity to entry inhibitors have to be sought outside the gp120 V3 and gp41 HR1 regions.
In addition to the acquisition of basic knowledge regarding HIV-related pathogenesis, studies on the evolution of R5 HIV-1 variants with improved binding abilities that result in enhanced viral fitness may prove to be important for the identification of the mechanisms that determine baseline susceptibility to entry inhibitors, and so aid efforts to design optimal treatment strategies.
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ACKNOWLEDGEMENTS |
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Received 14 April 2005;
accepted 12 July 2005.
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