Introduction
Productive infection of CD4+ T cells, in vitro, is often accompanied by the appearance of multinucleated giant cells, or syncytia [1,2]. It has been shown in HIV-1-infected cell cultures that cell-to-cell fusion and the formation of syncytia are induced via interactions between the viral envelope glycoprotein on HIV-infected cells and CD4 molecules on the surface of other cells [2]. Syncytia is one of the major causes of HIV-1-induced cell death, in vitro and possibly in vivo [3]. Clinical AIDS is characterized by opportunistic manifestations, in addition to the appearance of syncytium-forming HIV variants in approximately 50% of infected patients. These syncytium-forming variants differ from nonsyncytium-forming (NSI) variants in the V3 loop [4], are CD4+ cell tropic and are associated with a rapid CD4+ T-cell decline [5,6]. Thus, the syncytium-forming phenotype is a marker for HIV disease progression and the decline in CD4+ T-cell counts may be both a cause and an effect of CXCR4 prevalence [7]. Further, HIV-related neurocognitive disorders have a strong association with the presence of syncytium-inducing HIV-1 isolates and multinucleated giant cell formation [8].
HIV-induced syncytia have also been observed from a range of in-vivo tissues such as the mucosal surface of adenoids and tonsils of HIV-positive individuals [9,10]. In addition, Lewin-Smith et al. [11] also identified virus budding from the membrane of these giant cells within the colon, implying their potential to contribute the virus in circulation. Thus, overall, syncytia formation plays an integral role in HIV pathogenesis.
As syncytium formation includes fusion of a variety of leukocytes, it could potentially bring together diverse HIV variants from within a patient's quasispecies. Thus, in the setting of a coinfection with diverse intrasubtype or intersubtype HIV strains, syncytia may play an important role in the generation and dissemination of an array of HIV variants. A unique feature of retroviruses is that they carry two positive strands of viral RNA [12,13]. If the two strands of RNA are different (i.e. a heterozygous virion), a recombinant may emerge; thus, a prerequisite for recombination is a dually infected cell [13,14]. It is believed that via the fusion of multiple HIV-infected cells, a greater number of proviruses can be brought together, thereby facilitating recombination [14]; however, to date, biological proof of this is lacking.
Here, we have assessed the potential of syncytia in harboring multiple HIV-1 copies and variants and also virion production. A highly specific method to isolate individual syncytia that were generated in vitro from cells infected with two different HIV-1 subtypes, A and D, was developed. We also developed a method to conduct PCR and real-time PCR assays directly on singly isolated syncytia, in order to determine whether syncytia could harbor multiple HIV-1 variants, their proviral copy numbers and their capacity to act as precursors to recombinants.
Methods and materials
Viral strains
The viral strains used were both reportedly syncytia inducing. Subtype A-92Ug029 (X4) and subtype D-92Ug001 (R5X4) were obtained from the AIDS Research and Reference Reagent program (NIH, Bethesda, Maryland, USA). Stock cultures were generated in uninfected donor peripheral blood mononuclear cells (PBMCs) via standard culture procedures. The 50% tissue culture infective dose (TCID50) for each virus was determined using the Spearman-Karber method and converted to a multiplicity of infection.
Cell culture
Uninfected peripheral blood mononuclear cells were isolated using Ficoll-Hypaque (Amersham Pharmacia, Uppsala, Sweden) density gradient centrifugation from heparin-treated donor blood. Before HIV infection, cells were stimulated with 5 μg/ml phytohemagglutinin (PHA-P) for 2-3 days. Cells were maintained in RMPI (Life Technologies, Baltimore, Maryland, USA), which was supplemented with 10% fetal calf serum (CSL Biosciences, Parkville, Victoria, Australia), 20 U/ml IL-2 (Roche, Nutley, New Jersey, USA) and 100 units/ml each of penicillin and streptomycin (CSL Biosciences). Coculture was carried out over 14 days and it was replenished with fresh media every 3-4 days.
Dual cultures
Isolated donor PBMCs were infected with equal multiplicity of infection (MOI)(0.0002 each) of virus subtypes A and D. Cells were maintained for 6-10 days and monitored for syncytia production. When syncytia number was observed to be at a peak, generally 1+ syncytia per field of view, single syncytia were isolated (see Fig. 1).
Isolation of single syncytia
To remove and break cell clumps, cultures were filtered through a 40 μm cell strainer (Becton Dickinson, Franklin Lakes, New Jersey, USA). The flow-through was collected and again filtered through a 40 μm cell strainer, this time with a 20 μm net filter (Millipore, Billerica, Massachusetts, USA) inside the cell strainer to retain only syncytia of approximately greater than 20 fused cells, which were considerably larger than the infected single T cells. The culture was passed very slowly through this filter system and then washed extensively with phosphate-buffered saline (PBS) and bovine serum albumin (0.5 g/l) (BSA; Sigma, St Louis, Missouri, USA). The cell strainer and net filter were then inverted and placed in a Petri dish and the contents eluted. The dish was then placed under a microscope attached to a digital camera. Using the screen of the camera as a guide, individual syncytia were isolated into an Eppendorf tube by hand using a P20 pipette set to 10 μl. To ensure that only single syncytia were collected, and not cell clumps, each syncytium was viewed at high magnification to confirm the presence of a continuous cell membrane surrounding multiple nuclei. From each culture with 5 × 106 cells, we isolated only 10-30 syncytia. In addition, syncytia, seen in close proximity to other cells, were not collected to avoid contamination. Cell-free supernatants (10 μl) were also collected as controls. Samples were then heated at 75°C for 5 min to inactivate HIV and lyse syncytia. These syncytial lysates were then directly subjected to standard or real-time PCR.
Syncytia culture for virus isolation in vitro
Single syncytia were collected, as described above and shown in Fig. 1, and transferred to wells in a 96-well culture plate (Becton Dickinson). In addition, 10 μl aliquots of cell-free supernatants were collected as controls. To each well, 2 × 105 healthy donor PBMCs were added and cultured for 14 days. On day 7, 100 μl of the culture was removed and stored for p24 testing and 100 μl fresh media added. On day 14, the culture was terminated and the supernatant stored for p24 antigen testing.
Long-term cultures to examine the emergence of HIV-1 intersubtype recombinants
Additional dual cultures of HIV-1 strains 92Ug029 and 92Ug001 were established in triplicate, again with equal MOIs of each strain. These cultures were maintained for 28 days, with freshly stimulated PBMCs being added on a weekly basis and partial media changes every 3-4 days. A small number of cells were also collected every 3-4 days for DNA extraction, which was performed using the QIAGEN blood DNA mini kit (QIAGEN, Doncaster, Victoria, Australia). This proviral DNA was used in multiplex real-time PCR assays to determine intersubtype recombination events.
P24 enzyme-linked immunosorbent assay testing
Extracellular HIV-1 p24 antigen was measured in culture supernatants, seeded with single syncytia, using the p24-specific enzyme-linked immunosorbent assay (ELISA; Beckman-Coulter, Fullerton, California, USA). Supernatants tested were diluted 1: 2 with media prior to p24 testing and measured using an ELISA reader.
Standard polymerase chain reaction
PCR was conducted by adding 2.5 units of Taq polymerase, 1 mol/l Tris-HCl, 5 mmol/l KCl and 0.1% TritonX-100 (Promega, Madison, Wisconsin, USA), 2.5 mmol/l MgCl2, 200 μmol/l dNTPs (Promega), and 0.4 μmol/l of each primer with water to a final volume of 40 μl to isolated single syncytia. Env gp120-specific primers (forward: ED5 5′-ATGGGATCAAAGCCATGTG-3′; reverse: V2B 5′-GGAATTGGCTCAAAGGATACCTTT-3′) were effective in amplifying both subtypes used as input viral strains in the infection assay. To ensure syncytium lysis and maximum DNA yield, an additional cycling step of 95°C for 3 min and then 55°C for 3 min for three cycles was added [15]. Following this, 35 cycles of standard PCR were conducted at 95°C for 15 s, 55°C for 30 s and 72°C for 45 s. The positive samples were then cloned and sequenced.
Cloning
Cloning was performed as described elsewhere [16] using the pGEM-T easy vector system II (Promega) according to the manufacturer's instructions. The plasmids were transformed into Escherichia coli competent cells (ECOS cells) and grown overnight, with X-gal and isopropyl-b-D-thiogalactopyranoside (IPTG). White colonies were screened for the gene insert by quick lysis of the cells for 5 min at 95°C followed by PCR using only 25 cycles. Positive samples were then purified using a PCR purification plate on a vacuum manifold (Millipore).
Sequencing reaction
Purified PCR products were sequenced using the ABI Prism Big-dye Terminator Cycle Sequencing Ready Reaction kit (Version 3.1) on an ABI 377 automated sequencer.
Phylogenetic analysis of HIV-1 env clones derived from the single syncytia
Sequences derived from the clones in the HIV-1 env region from single syncytia were aligned against the known sequences of the two input strains, and an HIV subtype C strain which was used as an outgroup. CLUSTAL-W [17] was used to perform the alignment, and a neighbor-joining tree was constructed using the Kimura 2 parameter model in the PAUP* program [18].
Real-time polymerase chain reaction
Real-time PCR was performed using the Corbett Rotor-Gene 3000, using a dual-labeled fluorescent probe. The reaction mix of 25 μl total volume consisted of 1× RealMasterMix Probe, which included 1 U Taq, 5 mmol/l magnesium acetate and 0.4 mmol/l dNTPs (Eppendorf, Westbury, New York, USA), 50 nmol/l forward primer, 300 nmol/l reverse primer, 50 nmol/l probe, 10 μl of input DNA/single syncytia (in PBS and BSA) and water to 25 μl. The primers used were adapted from the study by Hoelscher et al. [19], to bind both subtypes (forward primer: MH-GAG 3.1:5′-RGTCAGCCAAAATTAYCCTATAG-3′ and reverse primer: MH-GAG 4.1 5′-GGGCTACACATGCCTGTGTRCC-3′). The probe sequence was derived from a conserved region between the primer sites as follows: 5′-FAM-TTTCAGCCCAGAAGTAATACCCATGTT-BHQ-1-3′. The real-time PCR reaction was as follows: 95°C for 1 min, 45 cycles of 95°C for 10 s, and then 60°C for 45 s, during which time the fluorescence data were collected.
All real-time PCR amplifications were quantitated by comparison with standards of known copy numbers. Standards were purified PCR products generated from plasmid DNA, which spanned slightly greater than the region of interest, and copy number was approximated by limiting dilution PCR [20]. The data were then analyzed using the Corbett Rotor gene 6 software.
Multiplex real-time polymerase chain reaction
Ten microliters of DNA extracted from long-term cultures was used in multiplex real-time PCR assays for the HIV-1 gag and gp41 regions, with subtype-specific primers and dual-labeled probes (see supplementary data). The assays were optimized and 0.5× Q-solution (QIAGEN) was added to each reaction, which increased the specificity of the primer-probe sets, to avoid crossreactivity. Primer-probe concentrations were also optimized to minimize competition within the assays. Other reaction conditions were as outlined above. The limits of detection for each species were within the same order of magnitude, down to 10 copies for each subtype within the gag region and 50-80 copies for A and D for the gp41 region. The subtype-specific primers and probes utilized are shown in Table 1.
Results
Isolation of single syncytia
The protocol, which we developed to isolate single syncytia from PBMC cultures, proved highly successful. The introduction of an initial filtration step proved useful in removing and dismantling cell clumps, which simplified the identification and collection of individual syncytia. The addition of BSA also improved the efficiency of collection, as the cells remained in suspension rather than becoming adherent, which also increased the overall recovery of syncytia from each culture. On an average, 20-60 individual syncytia were collected from a culture initiated with 5 × 106 PBMCs.
Viral isolation from syncytia in vitro
In order to confirm that the syncytia produced could act as a source of new infectious virus, single syncytia were isolated and seeded into a 96-well plate. Uninfected cells from HIV-negative healthy donors were then added and extracellular p24 antigen production in the culture supernatant was used as a measurement of virus replication in vitro, and viral turnover was monitored on days 7 and 14 after infection. Of 16 cultures infected with single syncytia, three had detectable p24 antigen production. To confirm that the infection was a direct result of infectious virus originating from the syncytia and not residual virus from the supernatant, cell-free controls were included. All of these controls tested negative for p24 antigen confirming that no cell-free virus was carried over (Fig. 2).
The nature of HIV variants in syncytia: polymerase chain reaction amplification of viral populations
To minimize the number of processing steps and loss of collected syncytia, we subjected individual syncytia directly to a modified PCR protocol, which circumvented the need for DNA extraction. The success rate varied from 70 to 100%. Cell-free controls were included to confirm that the generated PCR products did not arise from DNA in the supernatant. In each case, these controls tested negative by PCR. Ten PCR products from single syncytia were randomly chosen and multiple clones of the HIV env gene (HXB2 position 6591-6863) were generated and sequenced. On the basis of the sequence alignments and phylogenetic tree analysis of multiple clones derived from these syncytia, four showed the presence of both subtypes A and D within the single syncytia (Fig. 3 and Table 2).
Real-time polymerase chain reaction and assessment of proviral copy numbers within single syncytia
Syncytia collected from in-vitro HIV-infected cells of five separate healthy donor PBMCs were used to calculate proviral copies using real-time PCR. Each culture was established using equal MOIs (0.0004-0.0002) each of subtype A and subtype D viruses. Five to eight days after infection, single syncytia were individually collected, heat inactivated and then subjected directly to real-time PCR. The frequency of syncytia testing positive in the real-time PCR assay ranged from 20 to 70%. Syncytia had the tendency to adhere to pipette tip, thereby inhibiting their proper ejection into the collection tube, which may have contributed to some of the failures observed in our real-time PCR assays. Second, sometimes syncytia lyse upon contact with the pipette tip and can contribute to failed PCR results. Standards with known HIV-1 copy numbers were used to calculate the approximate number of proviral copies within each syncytium. Analysis revealed that the proviral copies varied widely between syncytia, ranging from approximately three copies to as high as 2275 copies per syncytium (Table 3).
For the real-time PCR experiments, a dual-labeled probe was used to detect the HIV-1 proviral DNA within single syncytia, as real-time PCR using Sybergreen was shown to be significantly hindered by the addition of as little as 3 μl of PBS in a total reaction volume of 25 μl. This could be attributed to the effect of salts on the binding and fluorescence capability of Sybergreen [21]. Tests with the dual-labeled probe indicated that PBS had negligible effect on the reaction.
Real-time analysis of long-term cultures
In dual HIV-1 subtype A and D cultures, a substantial number of large syncytia were observed 4-10 days after infection. A multiplex real-time PCR assay was then used on DNA extracted from cells of long-term cultures to compare the proportion of subtypes A and D across the gag and gp41 regions over the culture period. Subtype A gag and gp41 regions were detected throughout the entire culture period. However, for the subtype D viral strain, the gag region was no longer detectable after 21 days of culture, although the gp41 region was still present (Fig. 4). Further analysis of the assay system confirmed that within the multiplex gag reaction, an excess of subtype A did not hinder the detection of low copy numbers of subtype D, and the sensitivity of detection for each subtype within the gag region was actually greater than that within the gp41 region (approximately 10 and 80 copies, respectively). Thus, it is likely that the subtype D gag region was lost through recombination and the D gp41 region was retained in an A/D recombinant.
Discussion
Previous studies [3,22-25] have focused on the biological properties of multinucleated giant cells. These cells can significantly contribute to cell death and virus production in vitro [3]. In vivo, syncytium formation is known to contribute toward rapid HIV disease progression [26]. It is believed that HIV-induced syncytia may aid the generation of heterozygous virions [14], although the evidence to attest to this belief has been lacking. As the prevalence of dual infections and intersubtype recombinants is rising [27,28], it is important to understand underlying mechanisms of recombination.
To demonstrate that syncytia have a potential greater than single cells to act as a 'mating ground' as suggested by Burke [14], several points were considered in our study: syncytia must be able to produce viable virus; they must be able to carry at least two distinct viral variants vital for generation of heterozygous virions; and they must have a significantly larger proviral copy number than single cells. The most notable features of our study are the successful development of a system to isolate single syncytia and culture HIV from these, the demonstration that single syncytia can simultaneously harbor two distinct HIV-1 subtypes and that syncytia can harbor greater HIV-1 proviral copies than single cells. Together, our results provide experimental evidence that syncytia not only bring diverse HIV subtypes together but are also potential facilitators of intersubtype recombination.
In long-term dual cultures with the same viral strains, we were able to show some evidence of A/D intersubtype recombinants emerging. The loss of the subtype D gag region was observed after 21 days of culture; however, the subtype D gp41 region was still easily detected, indicating that a recombinant with a subtype A gag and a subtype D gp41 may have emerged. The identification of recombinants in cultures that had a large number of syncytia indicates that they may have played an important role in this process; however, the contribution of single cells to recombination cannot be excluded.
A study by Sylwester et al. [3] previously demonstrated virus budding from the surface of syncytia and initiated new HIV-1 cultures from single syncytia. We wished to confirm these results and show that the virus produced by single syncytia in our system was capable of initiating an infection in vitro. We were able to demonstrate the production of the HIV protein p24 antigen in three (of 16) cultures that were infected with single syncytia. Although this success rate was relatively low, the underlying reasons contributing to partial failure have been outlined above. Nonetheless, these results still indicate that viable syncytia can produce infectious virus.
In our system, single syncytia isolation proved to be very effective and highly specific; however, the use of these cells for culture and PCR was achieved with varying degrees of success. It is expected that all syncytia should have resulted in a successful PCR amplification of the input viruses; however, there are several reasons why this may not have always occurred. First, many younger syncytia appeared to be quite fragile and had a tendency to lyse rapidly during the collection process. Second, it is likely that syncytia retained in the pipette tip failed to be ejected because of their adherent nature. Alternatively, it is likely that some syncytia harbored a smaller number of proviral copies, which were below the limit of detection of assay. Even so, our system could successfully isolate single syncytia harboring two HIV subtypes.
Dang et al. [29] have shown that dual infections of cells occur at a higher frequency than that predicted by chance, in both direct and cell-mediated infections, which may contribute to the high rate of HIV recombination. However, syncytia may play a larger role in the generation of recombinants. In support of this, we were able to show that syncytia can bring together multiple HIV subtypes and can harbor a wide ranging number of proviral copies, which could be far in excess of what is seen in single cells. Jung et al. [30] used in-situ hybridization to estimate the number of proviral copies from single splenocytes from infected patients and found on average three to four copies, with a maximum of eight per cell. Studies [31,32] of infected PBMCs also concluded that the proviral copy numbers were likely to be small and close to one per cell.
It is also important to mention that our assay detects all forms of viral cDNA, integrated forms as well as circular, and unintegrated linear forms and thus values for copy number may be an overestimation of the true proviral copies that are able to give rise to subsequent progeny virus within the cell. A study [33] done in activated PBMCs also found that after 48 h of infection, approximately one-quarter of the total viral DNA within single cells was integrated, and another suggested that unintegrated forms were capable of producing some viral gene transcripts [34]. An in-vitro study [35] in a T-cell line detecting integrated and unintegrated forms was able to demonstrate copy numbers of up to 100 in total; however, again this figure is still lower than our copy number estimates for many single syncytia. Therefore, the finding of syncytia with copy numbers many times greater than those reported for single cells shows that syncytia are likely to be the result of fusion of many infected as well as uninfected cells. This goes against the previous assumption that syncytia arise from the fusion of one infected cell with other uninfected cells, which is likely to have arisen because of the observation that HIV infection leads to a downregulation of its cell surface receptors CD4, CCR5 and CXCR4 [36-38]. However, syncytia formation may begin to occur before complete downregulation has occurred, or fusion could still occur with residual receptors remaining on the cell surface.
The present study was conducted in vitro, in activated cells, and therefore is a partial representative of an in-vivo setting. It is possible that cells within the lymph node could behave in a similar fashion. Studies by Pope et al. [39], which are believed to closely mimic an in-vivo infection, have examined the formation of syncytia between dendritic cells and quiescent T-cells. Syncytia between these cells can bring together transcription factors that are believed to greatly enhance viral replication. Thus, our study provides yet another explanation why syncytia formation can be valuable for virus production, as they have a far greater potential than a single cell to give rise to heterozygous virions and therefore will likely contribute to recombination and consequently an overall increase in viral diversity.
Acknowledgements
M.C.S is grateful to the University of Sydney for the Australian Postgraduate Award for the PhD work, The Millennium Foundation for the top-up grant and a grant from the Retroviral Genetics Lab. The authors are grateful to the National Institutes of Health (NIH) Reagent Program, Bethesda, Maryland, USA for providing gratis reagents and viral strains used in this study.
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