Virology Division, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK1
School of Biological and Applied Sciences, University of North London, 166220 Holloway Road, London N7 8DB, UK2
Department of HIV and Genitourinary Medicine, The Guys, Kings and St Thomas School of Medicine, Kings College Hospital, Denmark Hill, London SE5 9RS, UK3
Author for correspondence: Natalie Zheng. Fax +44 208 906 4477. e-mail nzheng{at}nimr.mrc.ac.uk
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Abstract |
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Introduction |
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We have reported isolation of HIV-1 from LTNP, at high titre, in the herpesvirus saimiri-immortalized T cell (HVS T cell) line, CN-2 (Vella et al., 1999a ). CN-2 was immortalized from CD8-depleted, HIV-seronegative donor PBMCs and shown to express CD4, CD45RO, CD26, CD7, CCR5 and CXCR4 at the cell surface, reminiscent of activated memory T cells, which are targeted by HIV-1. However, several studies have reported the loss of genetic polymorphism present in HIV-1 quasispecies after isolation in T cell lines and PBMCs and the phrase to isolate is to disturb has been used (Meyerhans et al., 1989
; von Briesen et al., 1990
; Kusumi et al., 1992
). Alterations in biological characteristics, such as sensitivity to neutralizing antibodies between viruses grown in PBMCs and those grown in macrophages or T cell lines, have also been reported (Sawyer et al., 1994
; Willey et al., 1996
). To address such problems in relation to HIV-1 isolation in HVS T cells, viruses isolated from two patients in PBMCs and four HVS T cell lines were characterized phenotypically.
For HIV-1 genotypic analyses the env gene was selected as it encodes glycoproteins that are crucial for virus entry and are major determinants of cell tropism (reviewed in Douglas et al., 1997 ). Binding sites for host cell receptors reside on gp120 whilst membrane fusion components are contained in gp41. In addition, the glycoproteins are major targets of the host immune response and the only ones to which virus-neutralizing antibodies are raised. Complete env genes were amplified and cloned directly from patient DNA and plasma RNA extracts and compared with those in viruses produced by their respective culture isolates. Additionally, this approach supplied information on three HIV-1 regulatory genes (vpu, tat and rev), the translation products of which can have profound effects on virus replication.
PBMCs and HVS T cells were shown to have similar properties by selecting for viruses that would replicate in these two cell types only, with an NSI phenotype, and individual PBMC/HVS T cell virus pairs had similar neutralization profiles. Sequencing studies showed that both cell types selected similar variants from the virus quasispecies and, whilst selection was specific for individual patient/virus samples, all virus isolates displayed the common characteristic of carrying increased negative charge across their glycoprotein V2 domains compared with sequences derived directly from the patient samples. These results indicate that HVS T cells are a viable alternative to PBMCs for the isolation of HIV-1.
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Methods |
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A panel of 11 cell lines were used in cell tropism studies. These were the T cell lines MT-2, PM-1, JM, H9, C8166, Molt-4-clone8, Jurkat, Jurkat-tatIII and CEM-SS, the monocytoid line U937 and the human myeloid leukaemia cell line HL60. The HVS T cell lines CN-1 and CN-2 (Vella et al., 1999a ), AC (Vella et al., 1997
) and CP-1 (Vella et al., 1999b
) were used in virus isolation studies. The CD8-depleted, HIV-1 seronegative, donor PBMC pool has been described (Vella et al., 1999b
). CN-1 and CN-2 came from the same blood donation, provided by an HIV-1 seronegative donor (HLA class II haplotype: DRB1*0401, DRB1*0701, DRB4*0103, DRB4*0101, DQB1*0303, DQB1*0301), which had been CD8-depleted and, for CN-1, enriched for CD45RO and CD7 by FACS sorting, prior to immortalization. Likewise, CP-1 was immortalized from the CD8-depleted donor PBMC pool used for isolation. Cells were cultured as described previously (Vella et al., 1999a
, b
).
Mega-glioblastoma U87 cells stably expressing human CD4 and one human chemokine receptor (CCR1, CCR2b, CCR3, CXCR4 or CCR5) were employed in co-receptor usage assays (Bjorndal et al., 1997 ). The cells were grown in DMEM (Gibco) containing 15% FBS with 300 µg/ml G418. To select for co-receptor expression, cultures were supplemented with 1 µg/ml puromycin.
Antibodies.
Human plasma/sera and a panel of monoclonal antibodies (mAbs) were used to compare the sensitivity of PBMC and HVS T cell virus isolates to neutralization. Plasma samples were obtained from five LTNP (CW010, CW012, CW031, CW037 and CW082; Vella et al., 1999a ). V3 loop-binding mAbs were ARP3023 and ARP3056 and those recognizing the CD4 binding site were ARP388/389, ARP3054 and ARP3065.
Virus isolation and titration.
Freshly prepared patient PBMCs were co-cultivated with equal numbers of HIV-1 seronegative donor PBMCs that had been stimulated with PHA for 34 days. Subsequently, patient PBMCs (CW010 and CW012), which had been stored in liquid nitrogen for 3 months, were co-cultivated with equal numbers of HVS T cells. Production of virus was monitored with commercial p24-ELISA kits (Abbott). Samples containing the peak p24 concentrations were used to determine the biological and genotypic phenotypes of the rescued viruses. The 50% tissue culture infective dose (TCID50) of primary isolates rescued in either PBMCs or HVS T cells was determined on the freshly stimulated PBMC pool as described (Vella et al., 1999b ).
Virus neutralization assay.
The neutralization sensitivity of PBMC and CN-2 isolates was compared in both PBMCs and CN-2 using 7 day microneutralization assays with a constant virus challenge of 100 TCID50 against 1:5 dilutions of heat-inactivated serum/plasma. mAbs were used at a concentration of either 5 µg/ml (ARP3023, ARP388/389) or 10 µg/ml (ARP3056, ARP3065). For the assay, 50 µl aliquots of virus were mixed with 50 µl plasma/serum or mAb in duplicate in 96-well flat-bottomed microtitre plates and incubated at room temperature for 1 h. Freshly stimulated pooled PBMCs or CN-2 cells in exponential phase growth were then added (4x104 cells in 100 µl) and incubated overnight. Residual p24/Ab was removed with extensive washes, and plates incubated for a further 6 days, after which virus production was measured by p24-ELISA. Virus controls (virus plus cells alone), cell controls (cells plus antibody alone) and neutralization of tissue-culture-adapted strains of HIV-1, GB8 and JRFL, were included. The neutralization titre (% inhibition of p24 production) was determined at 7 days post-infection (p.i.) using the formula: % inhibition=mean OD of virus controls-(mean OD of virus+Abs)/mean OD of virus controls x100.
Neutralization by serum/plasma and mAbs (at the concentrations used) was considered positive when an inhibition of 70% or greater was observed.
Cell tropism and co-receptor usage.
The appropriate T cell line and PHA-stimulated PBMCs (5x103 cells each) were inoculated in duplicate with 50 µl of virus-containing culture supernatant, in 96-well round-bottomed plates, and incubated for 3 h at 37 °C. After washing three times with RPMI, the cells were resuspended in 200 µl of culture medium. Half of the culture supernatant was removed and replaced with fresh medium as required and syncytium formation was observed daily for 14 days. Supernatant was tested by p24-ELISA at 14 days p.i.
To determine the co-receptor usage of the viruses, 104 cells of each CD4+ U87 cell line expressing an HIV-1 co-receptor were inoculated with 200 TCID50 of virus (determined on PBMCs) and screened for infection as described (Dittmar et al., 1997 ).
Rescue of viral env genes.
Env genes were rescued from DNA extracts of uncultured patient PBMCs, and RNA extracts of plasma and culture supernatants. Viral RNAs were extracted from 100 µl of plasma or peak titre culture supernatants using the SDSproteinase K lysis/phenol/chloroform method and cDNA was synthesized using Primer NEFR (Table 1). For DNA and cDNA templates, two rounds of PCR were performed using primer pairs TATF/NEFR and FENV/RENV (Table 1
; Douglas et al., 1996
). Full-length env genes, with terminal XhoI and EcoRI sites, were cloned into the expression vector, pQ7. Clones that expressed full-length gp160, detected by Western blot, were selected for sequencing with a panel of primers (Table 1
). ABI 377 sequencers were used with Big-Dye kits and sequences assembled using gap-4 (Bonfield et al., 1995
).
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Results |
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Co-receptor usage assays showed that all isolates from CW012 infected U87/CD4+/CCR5+ cells only, confirming their R5 status, while isolates from CW010 in addition infected U87/CD4+/CCR3+ cells showing that they were of R5R3 phenotype. These results are in agreement with the NSI phenotypes of the viruses.
Sensitivity to neutralization
A panel of mAbs, autologous and heterologous plasma were tested against paired PBMC and CN-2 virus isolates (Table 2). Although small differences in sensitivity between viruses isolated from the same patient were seen, the neutralization profiles were similar using 70% inhibition of p24 production as compared with the virus control as the cut-off. Plasma C012 neutralized its own isolates whilst plasma C010 did not. Only plasma C082 neutralized virus from CW010. Plasmas C031 and C037 neutralized virus from patient CW012. Neither anti-V3 nor anti-CD4 binding site mAbs neutralized virus from patients CW010 and CW012.
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Phylogenetic analysis was performed on sequences generated here together with those of reference strains in the Los Alamos HIV Database (http://hiv-web.lanl.gov). The nucleotide alignment spanned 2772 positions, encompassing entire env genes and retaining gaps introduced to improve the alignment (predominantly in regions encoding gp120 variable domains). A thousand trees were calculated and a consensus tree generated. All CW-derived viruses belonged to HIV-1 subtype B and individual patient env gene clones grouped in distinct clusters with 100% bootstrap values at their origins, indicative of no cross-contamination during virus isolation and env gene cloning (Fig. 2). Whilst phylogenetic distances were small, HIV quasispecies were apparent in all five patients and there was some suggestion of disparity between provirus- and virus-derived clones, notably for patients CW002 and CW048, though this may reflect the relatively low number of clones sequenced from each source. For all five patients, virus isolation in PBMCs (clones A; Fig. 2
) selected for env genes that were distinct from those present in vivo (proviral DNA and plasma RNA, clones D and P; Fig. 2
). Similarly, for CW010 and CW012, where virus isolation in HVS T cells was attempted, recovered env genes were most closely related to those derived from their respective PBMC isolates, with one exception (CW012H-533), which had a plasma-derived sequence (CW012P-002) as its closest relative. Overall, this suggests selection of relatively minor HIV variants from the in vivo quasispecies during virus isolation by the cell co-culture routes.
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HVS T cell isolation was performed for patients CW010 and CW012 and glycoprotein sequences recovered related most closely to those seen in their respective PBMC isolates (Fig. 2), which suggests that selection pressures are similar in activated HIV-1-seronegative donor PBMCs and HVS T cells. Overall, intra-patient glycoprotein homologies, for in vivo sampling and culture isolates, fell in the range 87·299·9%, whilst inter-patient homologies were 82·487·0% (not shown). CW010-derived sequences showed the greatest inter-group diversity throughout the glycoprotein and they were the only set of sequences to carry inter-group sequence substitutions at positions that have been shown to affect CCR5 binding (data not shown). This may relate to the R5R3 phenotype of CW010 isolates, as the amino acid substitutions observed would be expected to reduce binding to CCR5 (Rizzuto et al., 1998
). In addition, whilst most in vivo clones had the typical GPGR motif at the tip of the V3 loop (LaRosa et al., 1990
), CW010D-005 and all culture isolate clones contained a GPGS motif, which would result in a loss of positive charge (Fig. 3a
). The presence of positive charge on V3 loops, notably at positions 9 and 27 in our alignment, has been associated with the ability of HIV-1 to induce syncytia (Fouchier et al., 1992
). On this basis, all clones derived from CW010 isolates were predicted to have an NSI phenotype, whilst three of eight in vivo-derived clones (CW010D-007, CW010P-007 and CW010P-008), carrying positively charged residues in these two positions, might be derived from SI viruses. The isolate sequences were most closely related to an in vivo-derived DNA sequence (CW010D-005), confirming phylogenetic results, though sequence variation in the cytoplasmic tail of gp41 was significant (data not shown), possibly indicating the presence of env gene recombinants in the quasispecies.
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PBMC isolation of HIV-1 from the other three patients (CW037, CW048 and CW002) showed individual selection patterns compared with sequences derived from in vivo samples but again all clones were predicted to be of NSI phenotype based on their V3 sequences (Fig. 3a). Since the selection profiles for virus isolation from the five patient samples were different, it was difficult to predict which of the substitutions might be important in HIV-1 isolation by cell co-culture. It is well established that physico-chemical properties are potentially useful in predicting which regions of aligned protein subfamilies are important in determining function (Caffrey et al., 2000
). The physico-chemical properties (hydrophobicity, volume and charge) were calculated by domain (defined in Douglas et al., 1997
) for each patients groups of in vivo- and isolate-derived glycoprotein clones. Students t-test was applied to monitor the significance of alterations observed. The significant difference profiles varied dramatically between patients, with CW037 showing the least significant differences (CW037<CW012<CW048<CW002<CW010; results not shown). However, when considering in vivo- and isolate-derived physico-chemical properties across the five patient samples, only the V2 domain showed significant and consistent trends in terms of increases in negative charge in virus isolates (Fig. 3b
).
The env gene contains coding information for three other genes. The vpu gene sequence overlaps the region encoding the signal peptide of the HIV-1 glycoprotein and the second exons of tat and rev genes are contained within the gp41 cytoplasmic tail coding region. The translated sequences for these three proteins did not show any remarkable features compared with sequences reported in the Los Alamos Database but did show the individuality of HIV-1 strains infecting each patient. All vpu reading frames were open and terminated at the usual position. The majority of tat exons terminated after encoding 29 amino acids but four clones from CW012 (D-751, D-776, P-002, H-533) were truncated prematurely, whilst another (A-039) showed an amino acid extension due to mutation of the normal stop codon. Similarly, the majority of rev exons terminated after encoding 91 amino acids except for eight clones from CW012, which contained a three amino acid extension (ECC) due to mutation of the normal stop codon. Two further clones, CW048P-082 and CW037D-364, contained premature terminations. It has been shown that fully functional and attenuated rev genes can persist in HIV-1-infected, asymptomatic individuals (Iversen et al., 1995 ). Generally there was high similarity between sequences derived directly from patient samples (DNA/plasma) and those of their respective HIV-1 isolates.
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Discussion |
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Virus isolates from CW010 and CW012 could be passaged in PBMCs only and all displayed an NSI phenotype. Furthermore, for CN-2/PBMC isolate pairs, the virus neutralization profiles were similar (Table 2). This suggested similar selection following isolation in PBMCs and HVS T cell lines. Env gene sequencing showed there to be selection following isolation for sequences different to those present in the in vivo (provirus and virus) situations. Whilst these selections involved different amino acid positions and regions of the glycoproteins for each patients isolates, the selections were similar in PBMCs and HVS T cell lines. Similarly, for patients where PBMC isolation only was performed, individual selection patterns were observed. In comparing the isolation patterns for all five patients, only the V2 domain showed a significant and consistent trend in terms of increases in negative charge compared with the in vivo situations. This observation supports the NSI phenotype of the CW010 and CW012 isolates, as it has been shown that SI isolates carry more positive charge in V2 compared with NSI isolates and small changes in V2 can affect virus tropism (Cornelissen et al., 1995
; Koito et al., 1995
; Vella et al., 1999c
). That this domain has important structurefunction determination properties is shown by the conservation of Asp 21 (Fig. 3b
), which is important for virus replication, and the observation that length differences in V2 modulate exposure of the CD4 binding site and the immunoreactivity of gp120 (Wang et al., 1995
; Fox et al., 1997
). These properties probably account for V2 being targeted by HIV-neutralizing antibodies (Shotton et al., 1995
; Wu et al., 1995
).
The translation products of the three accessory gene reading frames (vpu, tat and rev) were studied for evidence of selection following virus isolation. Vpu has two biological functions, down-regulation of CD4 and enhancement of virus release from infected cells (Miller & Sarver, 1997 ). Tat and Rev are trans-acting nuclear regulatory proteins, which play essential roles in the HIV-1 replication cycle (Pollard & Malim, 1998
; Jeang et al., 1999
). All Vpu carboxy-termini were intact and whilst there were patient/virus strain-specific sequences, there was neither evidence for selection following virus isolation nor the presence of inactivating substitutions. Tat and Rev exon 2 were patient/virus strain-specific and only for CW010 was there evidence of selection following virus isolation. The majority of exons were intact and probably functional, as there were no remarkable amino acid substitutions compared with sequences presented in the database. Overall, the majority of expression-competent env genes rescued in the course of this study should be functional in terms of the overlapping vpu, tat and rev genes.
The results presented here show that there are no significant differences in biological phenotype between HIV-1 isolates rescued in HVS T cell lines and PBMCs. Therefore, given their more stable phenotype, HVS T cells are an equivalent/better host for rescuing primary HIV-1 isolates than PBMCs. From env gene sequencing studies, it was observed that both cell types selected for viruses carrying more negative charge in the V2 domains of their glycoproteins, a property that corresponds with the observed NSI phenotype of all isolates. The significance of this selection is unknown but it may represent a common selection for viral sequences that display an M-tropic/NSI phenotype, as has been observed in different individuals during seroconversion to HIV-1 (Zhu et al., 1993 ). Consequently, the selection seen following HIV-1 isolation in PBMCs/HVS T cells may reflect that occurring during natural infection.
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Acknowledgments |
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Footnotes |
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Received 16 November 2001;
accepted 5 February 2002.